Detection and diagnosis of smoking related cancers

ABSTRACT

Gene probes for specific regions of chromosome 3 (3p21.3) and chromosome 10 (10q22) have been found to be tools for the diagnosis and prognosis of smoking related cancers such as non-small cell lung cancer (NSCLC). For example, these probes can be used with fluorescence in situ hybridization (FISH), and used to stratify smokers into high and low risk groups, as well as determine a patients susceptibility to the development of smoking related cancers.

The current application claims priority to provisional application60/222,811 filed Aug. 4, 2000, herein incorporated by reference.

The government may owns rights to this invention pursuant to NCI, Dept.of Health and Human Services contract number N01-CN-85184.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to the fields of oncology, genetics andmolecular biology. More particular the invention relates to the use oftwo probes for regions of human chromosomes 3 and 10 that are highlypredictive of the development of neoplasia and progression of neoplasticevents.

II. Related Art

Lung cancer is one of the leading causes of cancer death in the world.The high mortality rate for lung cancer probably results, at least inpart, from the lack of standard clinical procedures for the diagnosis ofthe disease at early and more treatable stages compared to breast,prostate, and colon cancers. There is also extremely poor prognosisassociated with diagnosis of the disease, especially in advanceddisease. It is important that strategies to detect early stage lungcarcinoma or its precursors, such as atypical squamous metaplasia,dysphasia and carcinoma-in-situ in subjects at high risk be devised.

Cigarette smoking over a prolonged period of time is the most importantrisk factor in the development of lung and other smoking relatedcancers, with other risk factors including exposure to passive smoking,certain industrial substances such as arsenic, some organic chemicals,radon and asbestosis, ingestion of alcohol, radiation exposure fromoccupational, medical and environmental sources, air pollution andtuberculosis. Many of these factors greatly increase the risk ofdevelopment of lung and other smoking related cancers if they occur in aperson who is concurrently a smoker.

Genetic detection of human disease states is a rapidly developing field(Taparowsky et al., 1982; Slamon et al., 1989; Sidransky et al., 1992;Miki et al., 1994; Dong et al., 1995; Morahan et al., 1996; Lifton,1996; Barinaga, 1996). However, some problems exist with this approach.A number of known genetic lesions merely predispose to development ofspecific disease states. Individuals carrying the genetic lesion may notdevelop the disease state, while other individuals may develop thedisease state without possessing a particular genetic lesion. In humancancers, genetic defects may potentially occur in a large number ofknown tumor suppresser genes and proto-oncogenes.

The genetic detection of cancer has a long history. One of the earliestgenetic lesions shown to predispose to cancer was transforming pointmutations in the ras oncogenes (Taparowsky et al., 1982). Deletion andmutation of p53 has been observed in bladder cancer (Sidransky et al,1991). Numerous studies have shown deletions in the 3p region arerelated to lung and other smoking related cancers (Mitsudomi et al.,1996, Shiseki et al., 1996, Wistuba et al., 2000, Wu et al., 1998, andShriver et al., 1998).

Molecular studies (fluorescence in situ hybridization (FISH) forpolysomies, PCR for hypervariable markers (MI) and LOH, or specificmutations) have demonstrated that morphologically normal areas ofbronchial epithelium closest to the carcinomas frequently show the mostmolecular abnormalities (3p, 17p, 9p, 5q). In particular, the short armof chromosome 3 has been shown to frequently harbor deletions of allelesin several regions including 3p25-26, 3p21.3-22, 3p14 and 3p12. Theseregions are presumed to be the site of tumor suppressor genes, and lossof chromosome 3p allelles have shown to be an early event in lungtumorigenesis.

Chromosomal alterations in several cancers have been investigated, andfrequent LOH at chromosome 10 has been reported in a variety of cancers,including glioma, glioblastoma multiforme, prostate cancer, endometrialcancer, chondrosarcome, bladder cancer, malignant melanoma, andfollicular thyroid tumors ((Licciardello et al., 1989; Auerbach et al.,N. Engl J. Med., 265: 253-267, 1961; Voravud, et al., 1993; Feder etal., 1998; Yanagisawa et al., 1996; Thiberville et al., 1995;Papadimitrakopoulou et al., 1996; Zou et al., 1998; Brugal et al., 1984;Dalqueni et al., 1997; Muguerza et al., 1997).

Deletion rates of chromosome 3p are known to correlate with lung cancer.However, there is no current clinical method for the identifying apopulation of individuals who are at a high risk to develop lung cancersor upper airway primary or secondary cancers. A technique fordetermining the risks of developing these cancers would be of greatvalue for the ability to limit exposure to additional environmental riskfactors and to know when additional tests, supplements, or treatmentsare appropriate.

In various studies, chromosome deletions have been studied asidentifiers for lung cancers. For example, Shiseki et al, (1996)analyzed 85 loci on all 22 autosomal chromosomes to determine that theincidence of LOH on chromosome arms 2q, 9p, 18q, and 22 q in brainmetastases were significantly higher than that in stages I primary lungtumors. Mitsudomi et al. (1996) used PCR-based analysis for thedetection of LOH in non-small cell lung cancer. Multiple regions onchromosome 3p were observed to show that deletions of the 3p chromosomemay help to identify non-small cell lung cancer patients with a poorprognosis. Wistuba et al. (2000) used fifty-four polymorphic markersused to study the entire chromosome arm 3p and concluded that 3p alleleloss is nearly universal in lung cancer pathogenesis. Wu et al. (1998)studied 3p21.3 deletion using the probe, D3S4604/luca. Peripheral bloodlymphocytes of 40 lung cancer patients were observed to give theconclusion that lung cancer patients exposed to benzo[α]pyrene, a commonbyproduct of tobacco smoke, have frequent deletions in peripheral bloodlymphocytes. Shriver et al. (1998) studied lung cancer cell lines andidentified the human homolog of the L14 ribosomal protein gene, RPL14;deletion of RPL14 was shown to be related to the development of lungcancer. None of theses studies, however, are able to predict thesusceptibility of a patient to the development of lung cancer or topredict whether smokers and non smokers are at a high risk of developinglung or other smoking related cancers.

Because of the grim prognosis of lung cancer with a ten year survivalrate of <5% the only curable cancers are those diagnosed in the earlystages and treated surgically. There is a shift of interest towardsdiagnosis and study of early and preneoplastic states. Because earlydetection and effective chemoprevention therapy have potential to becurative, it is imperative to stratify the patients in clinical trials.These patients need to be monitored fore results of chemopreventiontherapy and also for predictions whether a particular preneoplasticlesion may progress.

SUMMARY OF THE INVENTION

The present invention provides probes located on chromosomes 3p21.3 and10q22 useful in the diagnosis and prognosis of cancers related tosmoking. In one embodiment, a method for identifying a subject at highrisk for the development, recurrence, or metastasis of cancer comprisingthe steps of (a) obtaining a test sample from a subject; (b) providing anucleic acid probe targeting RPL14, CD39L3, PMGM, or GC20; (c)contacting the probe with the test sample; and (d) analyzing DNA fromthe sample whereby aberrations in the hybridization of said probe tosaid DNA was compared to wild type DNA, indicating the risk for thedevelopment, recurrence, or metastasis of cancers.

More specifically the method identifies the risk for the development ofcancers. The cancer may be lung, upper airway primary or secondary, heador neck, bladder, kidneys, pancreas, mouth, throat, pharynx, larynx,esophagus, brain, liver, spleen, kidney, lymph node, small intestine,pancreas, blood cells, colon, stomach, breast, endometrium, prostate,testicle, ovary, skin, bone marrow and blood cancer. In preferredembodiments, the cancer is lung cancer. The test sample can include, butis not limited to, a surgical or biopsy specimen, paraffin embeddedtissue, frozen tissue, surgical fine needle aspirations, bronchialbrushes, bronchial washes, bronchial lavages, buccal smears, sputa,peripheral blood lymphocytes, esophageal brush, a fine needleaspiration, urinary specimens such as bladder washings and voided urine,and esophageal washes.

In one embodiment, it is provided that the subject can come from a groupcomprising smokers, former smokers, or non-smokers. In a similarembodiment, the test sample comes from said subject who has notpreviously been diagnosed with cancer.

It is a further embodiment of this invention that additional testing,agents or treatments may be performed after the risk for the developmentof said cancers has been analyzed. This includes, but is not limited to,a spiral CT-scan, cancer therapies and pharmaceutical treatments whichcan include radiotherapeutic agents, surgical treatment for removal ofthe cancerous growth, chemotherapeutic agents, antibiotics, alkylatingagents and antioxidants, biological modifying respidase drugs and otheragents. These agents and treatments can be used alone or in combinationwith other agents.

In certain embodiments, it is contemplated that FISH is used to measurethe aberrations in the particular loci. A unique 3p21.3 probe can befrom 1000 to 2000 base pairs or larger and used for detection in aregion of about 180,000 base pairs. The probe can be labeled with afluorophore, or more specifically digoxigenin. A specific 10q22 probecan be used in conjunction with the 3p21 probe. In certain embodiments,a control probe is used which can be labeled with a fluorophore, or morespecifically spectrum orange. The control probe is a chromosome 3 stablemarker or more specifically Centromere 3 (CEP 3).

In another embodiment, there is provided a method for identifying asubject at high risk for the development, recurrence, or metastasis ofcancer comprising: (a) obtaining a lung test sample from a subject; (b)providing a specific10q22 DNA probe; (c) contacting said probe with saidtest sample; and (d) analyzing DNA from said test sample, wherebyaberrations in the hybridization of said probe to said DNA is comparedto wild type DNA, indicating the risk for the development, recurrence ormetastasis of said cancers. More specifically the method identifies therisk of the recurrence or metastasis of cancers. In a furtherembodiment, the probe size is from 1000 to 2000 base pairs or larger,for detection in a region of about 200,000 base pairs. In an additionalembodiment, a specific 3p21 probe can be used with the 10q22 DNA probe.The control probe is a chromosome 10 stable marker, or more specificallyCentromere 10 (CEP10).

In another embodiment, there is provided a method for predicting theprogression or metastasis of non-small cell carcinoma and othercarcinoma in a subject comprising: (a) obtaining a test sample from asubject; (b) providing a RPL14, CD39L3, PMGM, or GC20 gene probe; (c)contacting said probe with said test sample; and (d) analyzing DNA fromsaid test sample.

In yet another embodiment, there is provided a method for predicting theprogression or metastasis of non-small cell carcinoma in a subjectcomprising: (a) obtaining a lung test sample from a subject; (b)providing a specific10q22 DNA probe; (c) contacting said probe with saidtest sample; and (d) analyzing DNA from said test sample.

In a further embodiment, there is provided a method for the staging lungof cancer in a subject comprising determining the deletion distributionof the 3p21.3 region.

In one embodiment, there is provided a method of determining likelihoodof relapse or a new primary for a cancer subject comprising determininggenetic aberrations at chromosomal loci 3p21.3 or 10q22 in DNA ofbronchial tissue adjacent to tumor tissue from said subject, whereinabnormalities in DNA of said adjacent tissue correlate with relapse ofsaid cancer. The cancer can comprise lung cancer or more specificallynon-small cell carcinoma, adenocarcinoma, or squamous cell carcinoma. Aspecific gene probe may comprise RPL 14, CD39L3, PMGM, or GC20, or a10q22 DNA probe. The 10q22 probe lies adjacent to the PTEN gene which isfrequently involved non-small cell cancer. Both the 3p and the 10q probecan be used simultaneously. The test sample can be chosen from the sameor contralateral lung, and can consist of tumorous or nontumorousbronchial cells.

In yet another embodiment, there is provided a method of identifying anindividual to be segregated from a high risk environment comprising: (a)obtaining a test sample from a subject; (b) providing a gene probecontaining RPL14, CD39L3, PMGM, and GC20 genes and PTEN or a 10q22 DNAprobe, (c) contacting said probe with said test sample; and (d)analyzing DNA from said test sample, whereby said analysis is used toidentify an individual who is highly susceptible to the development oflung cancer and who should not be exposed to a high risk environment.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawings will be provided by the Office upon request and payment of thenecessary fee.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1—3p Relapse Status. 3p21 and 10q22 deletion rates in adjacentbronchial epithelial cells of patients with benign lung disease,patients who developed stage 1 non-small cell cancer that did notrelapse, and patients with stage 1 non-small cell cancer with relapsecompared to 95% Cl N3P. Open squares indicate that the subjects aresmokers, closed circles indicate that the subjects do not smoke(p<0.001).

FIG. 2—10g Relapse Status. 10q22 deletion rates in adjacent bronchialepithelial cells of patients with benign lung disease, patients whodeveloped stage 1 non-small cell cancer that did not relapse, andpatients with stage 1 non-small cell cancer with relapse compared to 95%Cl N10Q. Open squares indicate that the subjects are smokers, closedcircles indicate that the subjects do not smoke (p<0.001).

FIG. 3—Lung Cancer Tissues. Diagram of tissue demonstrating histogenesisof lung cancer.

FIG. 4.—Percentage of Tumor cells in Dilutions. Chart showing thepercentage of cells with 3p21.33 deletion detected by FISH relative tothe concentration of a dilution sample.

FIG. 5—Normal Metaphase Cells. Microscope images where normal cellstypically display 2 CEP3 (orange) signals and 2 3p21.33 (green) signalsand tumor cells display 3 CEP3 (orange) signals and 2 3p21.33 (green)signals.

FIG. 6—Normal Interphase Cells (Lymphocytes). Microscope images wherenormal cells typically display 2 CEP3 (orange) signals and 2 3p21.33(green) signals and tumor cells display 3 CEP3 (orange) signals and 23p21.33 (green) signals.

FIG. 7—Normal Bronchial Wash Cell. Microscope images where normal cellstypically display 2 CEP3 (orange) signals and 2 3p21.33 (green) signalsand tumor cells display 3 CEP3 (orange) signals and 2 3p21.33 (green)signals.

FIG. 8—Lung Cancer Cells. Microscope images where normal cells typicallydisplay 2 CEP3 (orange) signals and 2 3p21.33 (green) signals and tumorcells display 3 CEP3 (orange) signals and 2 3p21.33 (green) signals.

FIG. 9—Lung Cancer Cells. Microscope images where normal cells typicallydisplay 2 CEP3 (orange) signals and 2 3p21.33 (green) signals and tumorcells display 3 CEP3 (orange) signals and 2 3p21.33 (green) signals.

FIG. 10—10Q as a Predictor of Relapse. In a multivariate analysis,looking at the outcome in 96 patients, the deletion of 10Q in adjacentbronchial epithelial cells is a significant predictor of relapse.

FIG. 11—10Q as a Predictor of Long Term Survival. In a multivariateanalysis, looking at the outcome in 96 patients, the deletion of 10Q inadjacent bronchial epithelial cells is a significant predictor ofrelapse long term survival.

FIG. 12—Interval for Patients who are Relapse Free. The proportion ofpatients who are relapse free from 0 to 108 months for patients who havea N10q value>5 and N10q≦5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. The Present Invention

As stated above, deletions in the 3p21.3 and 10q22 regions of humanchromosomes 3 and the 10 have been shown to be associated with cancers.The present invention has shown these regions also to be predictive ofthe development of neoplasia and progression of neoplastic events. Inparticular embodiments, the inventors have developed novel DNA FISHprobes and tested them on patients at M.D. Anderson Cancer Center(MDACC) in early stage non-small cell lung neoplasms using archivaltissue from stage I non-small cell cancers.

The probes are used in the early detection of cancer and inchemopreventive studies as an intermediate biomarker. Until now therehave been no reports about the application of these DNA probes toparaffin embedded clinical tumor specimens using fluorescence in situhybridization (FISH) and microdissection. The FISH technique allows themeasurement of an average level of deletion of a gene in a tumor, aswell as the actual number and distribution of the gene in individual,morphological cells. The inventors propose that deletion distribution ofthe RPL14, CD39L3, PMGM or GC20 genes and 10q22 locus are useful as adiagnostic tool in determining the stage of lung cancer patients.

A. Smoking Related Cancers

The current invention is useful for the prognosis and diagnosis of lungcancers, which can be defined by a number of histologic classificationsincluding: squamous cell carcinomas such as squamous carcinoma; smallcell carcinomas such as oat cell carcinoma, intermediate cell typecarcinoma, combined oat and cell carcinoma; adenocarcinomas such asacinar adenocarcinoma, papillary adenocarcinoma, bronchioloalveolarcarcinoma, and solid carcinoma with mucus formation; large cellcarcinoma such as giant cell carcinoma and clear cell carcinoma;adenosquamous carcinoma; carcinoid; and bronchial gland carcinomas suchas adenoid cystic, and mucoepidermoid carcinoma. Diagnosis and prognosisof other smoking related cancers is possible with these probes. Squamouscell carcinoma of the head and neck has the same risk factors as lungcancer is hypothesized to have similar etiology (Shriver, 1998).Similarly, smoking is an etiological factor for cancer of the bladder,head, neck, kidneys, pancreas, and cancer of the upper airways includingcancer of the mouth, throat, pharynx, larynx, or esophagus.

B. Tumorgenesis

The deletions of various genes in tumor tissue has been well studied inthe art. However, there remains a need for probes that are significantfor detecting early molecular events in the development of cancers, aswell as molecular events that make patients susceptible to thedevelopment of cancer. Probes used for the staging of cancer are also ofinterest. The proposed sequence leading to tumorigenesis includesgenetic instability at the cellular or submicroscopic level asdemonstrated by loss or gain of chromosomes, leading to ahyperproliferative state due to theoretical acquisition of factors thatconfer a selective proliferative advantage. Further, at the geneticlevel, loss of function of cell cycle inhibitors and tumor suppressorgenes (TSG), or amplification of oncogenes that drive cellproliferation, are implicated.

Following hyperplasia, a sequence of progressive degrees of dysplasia,carcinoma-in-situ and ultimately tumor invasion is recognized onhistology. These histologic changes are both preceded and paralleled bya progressive accumulation of genetic damage. At the chromosomal levelgenetic instability is manifested by a loss or gain of chromosomes, aswell as structural chromosomal changes such as translocation andinversions of chromosomes with evolution of marker chromosomes. Inaddition cells may undergo polyploidization. Single or multiple clonesof neoplastic cells may evolve characterized in many cases by aneuploidcell populations. These can be quantitated by measuring the DNA contentor ploidy relative to normal cells of the patient by techniques such asflow cytometry or image analysis.

C. Prognostic Factors and Staging

At present, the most important prognostic factor regarding the survivalof patients with lung cancer of non-small cell type is the stage ofdisease at diagnosis. Small cell cancer usually presents with widespread dissemination hence the staging system is less applicable. Thestaging system was devised based on the anatomic extent of cancer and isnow know as the TNM system based on anatomical size and spread withinthe lung and adjacent structures, regional lymph nodes and distantmetastases. The only hope presently for a curative procedure lies n theoperability of the tumor which can only be resected when the disease isat a low sage, that is confined to the lung.

Occult Carcinoma TX NO MO Occult carcinoma with bronchopulmonarysecretions containing malignant cells but without other evidence of theprimary tumor or evidence of metastasis Stage 1 TIS NO MO Carcinoma insitu T1 NO MO Tumor that can be classified T1 without any metastasis tothe regional lymph nodes T1 N1 MO Tumor that can be classified T1 withmetastasis to the lymph nodes in the ipsilateral hilar region only T2 N1MO Tumor that can be classified T2 without any metastasis to nodes ordistant metastasis Stage II T2 N1 MO Tumor classified as T2 withmetastasis to the lymph nodes in the ipsilateral hilar region only StageIII T3 with an N or M Any tumor more extensive than T2 N2 with an T or MAny tumor with metastasis to the lymph nodes in the mediastinum M1 withany T or N Any tumor with distant metastasis

D. Grading of Tumors

The histological type and grade of lung cancer do have some prognosticimpact within the stage of disease with the best prognosis beingreported for stage I adenocarcinoma, with 5 year survival at 50% and1-year survival at 65% and 59% for the bronchiolar-alveolar andpapillary subtypes (Naruke et al., 1988; Travis et al., 1995; Carriagaet al., 1995). For squamous cell carcinoma and large cell carcinoma the5 year survival is around 35%. Small cell cancer has the worst prognosiswith a 5 year survival rate of only 12% for patients with localizeddisease (Carcy et al., 1980; Hirsh, 1983; Vallmer et al., 1985). Forpatients with distant metastases survival at 5 years is only 1-2%regardless of histological subtype (Naruke et al., 1988). In addition tohistological subtype, it has been shown that histological grading ofcarcinomas within subtype is of prognostic value with welldifferentiated tumors having a longer overall survival than poorlydifferentiated neoplasms. Well differentiated localized adencarcinomahas a 69% overall survival compared to a survival rate of only 34% ofpatients with poorly differentiated adenocarcinoma (Hirsh, 1983). The 5year survival rates of patients with localized squamous carcinoma havevaried from 37% for well differentiated neoplasms to 25% for poorlydifferentiated squamous carcinomas (Ihde, 1991).

The histologic criteria for subtyping lung tumors is as follows:squamous cell carcinoma consists of a tumor with keratin formation,keratin pearl formation, and/or intercellular bridges. Adenocarcinomasconsist of a tumor with definitive gland formation or mucin productionin a solid tumor. Small cell carcinoma consists of a tumor composed ofsmall cells with oval or fusiform nuclei, stippled chromatin, andindistinct nuclei. Large cell undifferentiated carcinoma consists of atumor composed of large cells with vesicular nuclei and prominentnucleoli with no evidence of squamous or glandular differentiation.Poorly differentiated carcinoma includes tumors containing areas of bothsquamous and glandular differentiation.

E. Development of Carcinomas

The evolution of carcinoma of the lung is most likely representative ofa field cancerization effect as a result of the entire aero-digestivesystem being subjected to a prolonged period of carcinogenic insultssuch as benzylpyrenes, asbestosis, air pollution and chemicals othercarcinogenic substances in cigarette smoke or other environmentalcarcinogens. This concept was first proposed by Slaughter et al. (1953).Evidence for existence of a field effect is the common occurrence ofmultiple synchronous for metachronous second primary tumors (SPTs) thatmay develop throughout the aero-digestive tract in the oropharynx, upperesophagus or ipsilateral or contralateral lung.

Accompanying these molecular defects is the frequent manifestation ofhistologically abnormal epithelial changes including hyperplasia,metaplasia, dysplasia, and carcinoma-in-situ. It has been demonstratedin smokers that both the adjacent normal bronchial epithelium as well asthe preneoplastic histological lesions may contain clones of geneticallyaltered cells. (Wistuba et al., 2000).

Liciardello et al. (1989) found a 10-40% incidence of metachronoustumors and a 9-14% incidence of synchronous SPTs in the upper and loweraero-digestive tract, mostly in patients with the earliest primarytumors SPTs may impose a higher risk than relapse from the originalprimary tumor and may prove to be the major threat to long term survivalfollowing successful therapy for early stage primary head, neck or lungtumors. Hence it is vitally important to follow these patients carefullyfor evidence of new SPTs in at risk sites for new malignanciesspecifically in the aero-digestive system.

In addition to chromosomal changes at the microscopic level, multipleblind bronchial biopsies may demonstrate various degrees ofintraepithelial neoplasia at loci adjacent to the areas of lung cancer.Other investigators have shown that there are epithelial changes rangingfrom loss of cilia and basal cell hyperplasia to CIS in most light andheavy smokers and all lungs that have been surgically resected forcancer. (Auerbach et al., 1961). Voravud et al. (1993) demonstrated byin-situ hybridization (ISH) studies using chromosome-specific probes forchromosomes 7 and 17 that 30-40% of histologically normal epitheliumadjacent to tumor showed polysomies for these chromosomes. In additionthere was a progressive increase in frequency of polysomies in thetissue closest to the carcinoma as compared to normal control oralepithelium from patients without evidence of carcinoma. The findings ofgenotypic abnormalities that increased closer to the area of the tumorsupport the concept of field cancerization. Interestingly there was noincrease in DNA content as measured in the normal appearing mucosa in aFeulgen stained section adjacent to the one where the chromosomes weremeasured, reflecting perhaps that insufficient DNA had been gained inorder to alter the DNA index. Interestingly a very similar increase inDNA content was noted both in dysplastic areas close to the cancer andin the cancerous areas suggesting that complex karyotypic abnormalitiesthat are clonal have already been established in dysplastic epitheliumadjacent to lung cancer. Others have also shown an increase in number ofcells showing p53 mutations in dysplastic lesions closest to areas ofcancer, which are invariably also p53 mutated. Other chromosomalabnormalities that have recently been demonstrated in tumors anddysplastic epithelium of smokers includes deletions of 3p, 17p, 9 p and5q (Feder et al., 1998; Yanagisawa et al., 1996; Thiberville et al.,1995).

F. Chromosome Deletions in Lung Cancer

Small cell lung cancer (SCLC) and non-small cell lung cancer commonlydisplay cytogenetically visible deletions on the short arm of chromosome3 (Hirano et al., 1994; Valdivieso et al., 1994; Cheon et al.,1993;Pence et al., 1993). This 3p deletion occurs more frequently in the lungtumor tissues of patients who smoke than it does in those of nonsmokingpatient. (Rice et al., 1993) Since approximately 85% lung cancerpatients were heavy cigarette smokers (Mrkve et al., 1993), 3p mightcontain specific DNA loci related to the exposure of tobaccocarcinogens. It also has been reported that 3p deletion occurs in theearly stages of lung carcinogenesis, such as bronchial dysplasia (Pantelet al., 1993). In addition to cytogenetic visible deletions, loss ofheterozygosity (LOH) studies have defined 3-21.3 as one of the distinctregions that undergo loss either singly or in combination (Fontanini etal., 1992; Liewald et al., 1992). Several other groups have found largehomozygous deletions at 3p21.3 in lung cancer (Macchiarini et al., 1992;Miyamoto et al., 1991; Ichinose et al., 1991; Yamaoka et al., 1990).Transfer of DNA fragments from 3-21.3-3p21.2 into lung tumor cell linescould suppress the tumorigenesis. (Sahin et al., 1990; Volm et al.,1989). These finding strongly suggest the presence of at least one tumorsuppressor gene in this specific chromosome region whose loss willinitiate lung carcinogenesis.

Cytogenetic observation of lung cancer has shown an unusual consistencyin the deletion rate of chromosome 3p. In fact, small cell lung cancer(SCLC) demonstrates a 100% deletion rate within certain regions ofchromosome 3p. Non small cell lung cancer (NSCLC) demonstrates a 70%deletion rate (Mitsudomi et al., 1996; Shiseki et al., 1996). Loss ofheterozygosity and comparative genomic hybridization analysis have showndeletions between 3p14.2 and 3p21.3 to be the most common finding forlung carcinoma and is postulated to be the most crucial change in lungtumorigenesis (Wu et al., 1998). It has been hypothesized that band3p21.3 is the location for lung cancer tumor suppressor genes. Thehypothesis is supported by chromosome 3 transfer studies, which reducedtumorigenicity in lung adenocarcinoma.

Allelotype studies on non-small cell lung carcinoma indicated loss ofgenetic material on chromosome 10q in 27% of cases. Studies ofchromosome 10 allelic loss have shown that there is a very highincidence of LOH in small cell lung cancer, up to 91%. (Alberola et al.,1995; Ayabe et al., 1994). A statistically significant LOH of alleles on10q was noted in metastatic squamous cell carcinoma (SCC) in 56% ofcases compared to non-metastatic SCC with LOH seen in only 14% of cases.(Ayabe et al., 1994). No LOH was seen in other subtypes on NSCLC.Peterson (1995) used paired samples of tumor and normal tissue to assessLOH. By micro-satellite polymorphism analysis, a high incidence of losswas found between D10s677 and D10S1223. This region spans the long armof chromosome 10 at bands q21-q24 and overlaps the region deleted in thea study of advanced stage high grade bladder cancers which demonstrateda high frequency of allele loss within a 2.5cM region at 10q22.3-10q23.1(Kim et al, 1996).

II. The 3p21.3 Gene Probes

A. Structural Features

Recently, the human ribosomal L14 (RPL14) gene (GenBank AccessionNM_(—)003973, SEQ ID NO: 1), and the genes CD39L3 (GenBank AccessionAAC39884 and AF039917; SEQ ID NO: 3), PMGM (GenBank Accession P15259andJ05073; SEQ ID NO: 5), and GC20 (GenBank Accession NM_(—)005875; SEQ IDNO: 7) were isolated from a BAC (GenBank Accession AC019204, hereinincorporated by reference) and located in the 3p21.3 band within thesmallest region of deletion overlap of various lung tumors. The RPL14gene sequence contains a highly polymorphic trinucleotide (CTG) repeatarray, which encodes a variable length polyalanine tract. Polyalaninetracts are found in gene products of developmental significance thatbind DNA or regulate transcription. For example, Drosophila proteinsEngraled, Kruppel and Even-Skipped all contain polyalanine tracts thatact as transcriptional repressors. Genotype analysis of RPL14 shows thatthis locus is 68% heterozygous in the normal population, compared with25% in NSCLC cell lines. Cell cultures derived from normal bronchialepithelium show a 65% level of heterozygosity, reflecting that of thenormal population.

B. Functional Aspects

Genes with a regulatory function such as the RPL14 gene (SEQ ID NO: 1),along with the genes CD39L3, PMGM, and GC20 (SEQ ID NOS: 3, 5 and 7) andanalogs thereof, are good candidates for diagnosis of tumorigenicevents. It has been postulated that functional changes of the RPL14protein (SEQ ID NO: 2) can occur via a DNA deletion mechanism of thetrinucleotide repeat encoding for the protein. This deletion mechanismmakes the RPL14 gene and attractive sequence that may be used as amarker for the study of lung cancer risk (Shriver et al., 1998). Inaddition, the RPL14 gene shows significant differences in allelefrequency distribution in ethnically defined populations, making thissequence a useful marker for the study of ethnicity adjusting lungcancer (Shriver et al., 1998). Therefore, this gene is useful in theearly detection of lung cancer, and in chemopreventive studies as anintermediate biomarker.

III. The 10q22 Gene Probes

A. Structural Features

The 10q22 BAC (46b12) is 200 Kb and is adjacent and centromeric toPTEN/MMAC1 (GenBank Accession AF067844), which is at 10q22-23 and can bepurchased through Research Genetics (Huntsville, Ala.). Alterations to10q22-25 has been associated with multiple tumors, including lung,prostate, renal, and endomentrial carcinomas, melanoma, and meningiomas,suggesting the possible suppressive locus affecting several cancers inthis region. The PTEN/MMAC1 gene, encoding a dual-specificityphosphatase, is located in this region, and has been isolated as a tumorsuppressor gene that is altered in several types of human tumorsincluding brain, bladder, breast and prostate cancers. PTEN/MMAC1mutations have been found in some cancer cell lines, xenografts, andhormone refractory cancer tissue specimens. Because the inventor's 10q22BAC DNA sequence is adjacent to this region, the DNA sequences in theBAC 10q22 may be involved in the genesis and/or progression of humanlung cancer.

B. Functional Aspects

Functional evidence for the presence of tumor suppressor genes on 10qhas been provided by microcell-mediated chromosomal transfer. Theresulting hybrid clones displayed a suppressed tumorigenic phenotypewith the inability to proliferate in nude mice and soft agarose.Sequence analysis of the PTEN/MMAC1 gene in lung cancer revealed a G toC substitution located 8 bp upstream of the coding region of exon1 andwhich seems to be a polymorphism, in 4 of the 30 cases of lung cancertested. Somatic mutations of the TPEN/MMAC1 gene were not identified inany of the tumors at the primary and metastatic sites of lung cancer,indicating that point mutations in the PTEN/MMAC1 gene are probably notan important factor in tumorigenesis and the progression of a majorsubset of lung cancers. Other more important tumor suppressor genes mustlie close to the PTEN/MMAC1 gene, in the vicinity of the inventors'10q22 BAC locus. Therefor, the 10q22 probe is useful in the furtherdevelopment of clinical biomarkers for the early detection of neoplasticevents, for risk assessment and monitoring the efficacy ofchemoprevention therapy in high risk former or current smokers.

IV. Nucleic Acids

The inventors' have identified the probes for the human chromosomeregion 3p21.3 and human chromosome region 10q22. In addition, it shouldbe clear that the present invention is not limited to the specificnucleic acids disclosed herein.

A. Probes and Primers

Naturally, the present invention encompasses DNA segments that arecomplementary, or essentially complementary, to target sequences.Nucleic acid sequences that are “complementary” are those that arecapable of base-pairing according to the standard Watson-Crickcomplementary rules. As used herein, the term “complementary sequences”means nucleic acid sequences that are substantially complementary, asmay be assessed by the same nucleotide comparison set forth above, or asdefined as being capable of hybridizing to a target nucleic acid segmentunder relatively stringent conditions such as those described herein.These probes may span hundreds or thousands of base pairs.

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that exemplary oligonucleotides of about 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 250, 500, 700, 722, 900, 992,1000, 1500, 2000, 2500, 2800, 3000, 3500, 3800, 4000, 5000 or more basepairs will be used, although others are contemplated. As mentionedabove, longer polynucleotides encoding 7000, 10000. 12000 bases andlonger are contemplated as well. Such oligonucleotides will find use,for example, as probes in FISH, Southern and Northern blots and asprimers in amplification reactions.

It will be understood that this invention is not limited to theparticular probes disclosed herein and particularly is intended toencompass at least nucleic acid sequences that are hybridizable to thedisclosed sequences or are functional sequence analogs of thesesequences. For example, a partial sequence may be used to identify astructurally-related gene or the full length genomic or cDNA clone fromwhich it is derived. Those of skill in the art are well aware of themethods for generating cDNA and genomic libraries which can be used as atarget for the above-described probes (Sambrook et al., 1989).

For applications in which the nucleic acid segments of the presentinvention are incorporated into vectors, such as plasmids, cosmids orviruses, these segments may be combined with other DNA sequences, suchas promoters, polyadenylation signals, restriction enzyme sites,multiple cloning sites, other coding segments, and the like, such thattheir overall length may vary considerably. It is contemplated that anucleic acid fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

DNA segments encoding a specific gene may be introduced into recombinanthost cells and employed for expressing a specific structural orregulatory protein. Alternatively, through the application of geneticengineering techniques, subportions or derivatives of selected genes maybe employed. Upstream regions containing regulatory regions such aspromoter regions may be isolated and subsequently employed forexpression of the selected gene.

B. Labeling of Probes

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, chemiluminescent, electroluminescent, enzymatic tag orother ligands, such as avidin/biotin, antibodies, affinity labels, etc.,which are capable of being detected. In preferred embodiments, one maydesire to employ a fluorescent label such as digoxigenin, spectrumorange, fluorosein, eosin, an acridine dye, a rhodamine, Alexa 350,Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, cascade blue, Cy2, Cy3, Cy5,6-FAM, HEX, 6-JOE,Oregon green 488, Oregon green 500, Oregon green 514, pacific blue, REG,ROX, TAMRA, TET, or Texas red.

In the case of enzyme tags such as urease alkaline phosphatase orperoxidase, calorimetric indicator substrates are known which can beemployed to provide a detection means visible to the human eye orspectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples. Examples of affinitylabels include but are not limited to the following: an antibody, anantibody fragment, a receptor protein, a hormone, biotin, DNP, or anypolypeptide/protein molecule that binds to an affinity label and may beused for separation of the amplified gene.

The indicator means may be attached directly to the probe, or it may beattached through antigen bonding. In preferred embodiments, digoxigeninis attached to the probe before denaturization and a fluorophore labeledanti-digoxigenin FAB fragment is added after hybridization.

C. Hybridization Conditions

Suitable hybridization conditions will be well known to those of skillin the art. Conditions may be rendered less stringent by increasing saltconcentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

V. Biomarkers

Various biomarkers of prognostic significance can be used in conjunctionwith the 3p21.3 or the 10q22 nucleic acid probes. These biomarkers couldaid in predicting the survival in low stage cancers and the progressionfrom preneoplastic lesions to invasive lung cancer. These markers caninclude proliferation activity as measured by Ki-67 (MIB1), angiogenesisas quantitated by expression of VEGF and microvessels using CD34,oncogene expression as measured by erb B2, and loss of tumor suppressergenes as measured by p53 expression.

Multiple biomarker candidates have been implicated in the evolution ofneoplastic lung lesions. Bio-markers that have been studies includegeneral genomic markers including chromosomal alterations, specificgenomic markers such as alterations in proto-oncogenes such as K-Ras,Erbβ1/EGFR, Cyclin D; proliferation markers such as Ki67 or PCNA,squamous differentiation markers, and nuclear retinoid receptors(Papadimitrakopoulou et al., 1996) The latter are particularlyinteresting as they may be modulated by specific chemopreventive drugssuch as 13-cis-retinoic acid or 4HPR and culminate in apoptosis of thedefective cells with restoration of a normally differentiated mucosa(Zou et al., 1998).

A. Tumor Angiogenesis by Microvessel Counts

Tumor angiogenesis can be quantitated by microvessel density and is aviable prognostic factor in stage 1 NSCLC. Tumor microvessel densityappears to be a good predictor of survival in stage 1 NSCLC.

B. Vascular Endothelial Growth Factor (VEGF)

VEGF (3,6-8 ch 4) an endothelial cell specific mitogen is an importantregulator of tumor angiogenesis who's expression correlates well withlymph node metastases and is a good indirect indicator of tumoragniogenesis. VEGF in turn is upregulated by P53 protein accumulation inNSCLC.

C. p53

The role of p53 mutations in predicting progression and survival ofpatients with NSCLC is widely debated. Although few studies imply anegligible role, the majority of the studies provide compelling evidenceregarding the role of p53 as one of the prognostic factors in NSCLC. Theimportant role of p53 in the biology of NSCLC has been the basis foradenovirus mediated p53 gene transfer in patients with advanced NSCLC(Carcy et al., 1980). In addition p53 has also been shown to be anindependent predictor of chemotherapy response in NSCLC. In a recentstudy (Vallmer et al., 1985), the importance of p53 accumulation inpreinvasive bronchial lesions from patients with lung cancer and thosewho did not progress to cancer were studied. It was demonstrated thatp53 accumulation in preneoplastic lesions had a higher rate ofprogression to invasion than did p53 negative lesions.

D. c-erb-B2

Similar to p53, c-erg-132 (Her2/neu) expression has also been shown tobe a good marker of metastatic propensity and an indicator of survivalin these tumors.

E. Ki-67 Proliferation Marker

In addition to the above markers, tumor proliferation index as measuredby the extent of labeling of tumor cells for Ki-67, a nuclear antigenexpressed throughout cell cycle correlates significantly with clinicaloutcome in Stage 1 NSCLC (Feinstein et al., 1970). The higher the tumorproliferation index the poorer is the disease free survival labelingindices provides significant complementary, if not independentprognostic information in Stage 1 NSCLC, and helps in the identificationof a subset of patients with Stage 1 NSCLC who may need more aggressivetherapy.

VI. Prognosis and Diagnosis of Cancers Using 3p21.3 and 10q22 GeneProbes

Alterations in the 3p21.3 and 10q22 loci are known to be associated witha number of cancers. More specifically, point mutations, deletions,insertions or regulatory perturbations relating to the 3p21.3 and 10q22loci may cause cancer or promote cancer development, cause or promotertumor progression at a primary site, and/or cause or promote metastasis.Other phenomena at the 3p21.3 and 10q22 loci include angiogenesis andtissue invasion. Thus, the present inventors have demonstrated thatdeletions at 3p21.3 and 10q22 can be used not only as a diagnostic orprognostic indicator of cancer, but to predict specific events in cancerdevelopment, progression and therapy.

A variety of different assays are contemplated in this regard, includingbut not limited to, fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNase protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR-SSCP.

Various types of defects are to be identified. Thus, “alterations”should be read as including deletions, insertions, point mutations andduplications. Point mutations result in stop codons, frameshiftmutations or amino acid substitutions. Somatic mutations are thoseoccurring in non-germline tissues. Germ-line tissue can occur in anytissue and are inherited.

A. Samples

One embodiment of the instant invention comprises a method for detectingvariation in the hybridization of the probes to DNA. This may comprisedetermining specific alterations in the expressed product, or may simplyinvolve detecting gross structural abnormalities. Such cancer mayinvolve cancers of the lung, upper airway primary or secondary cancer,bladder, urithial, head and neck, esophagus, kidney, pancreas, mouth,throat, pharynx, larynx, brain, liver, spleen, small intestine, bloodcells, lymph node, colon, breast, endometrium, stomach, prostate,testicle, ovary, skin, bone marrow, blood or other tissue.

In particular, the present invention relates to the diagnosis andprognosis of smoking related cancers. More particularly, the presentinvention relates to the diagnosis and prognosis of lung cancer whichincludes, but is not limited to: squamous cell carcinomas such assquamous carcinoma; small cell carcinomas such as oat cell carcinoma,intermediate cell type carcinoma, combined oat and cell carcinoma;adenocarcinomas such as acinar adenocarcinoma, papillary adenocarcinoma,bronchioloalveolar carcinoma, and solid carcinoma with mucus formation;large cell carcinoma such as giant cell carcinoma and clear cellcarcinoma; adenosquamous carcinoma; carcinoid; and bronchial glandcarcinomas such as adenoid cystic, and mucoepidermoid carcinoma.

The biological sample can be any tissue or fluid that contains nucleicacids. Various embodiments include paraffin imbedded tissue, frozentissue, surgical fine needle aspirations, cells of the skin, muscle,lung, head and neck, esophagus, kidney, pancreas, mouth, throat,pharynx, larynx, esophagus, facia, brain, prostate, breast, endometrium,small intestine, blood cells, liver, testes, ovaries, colon, skin,stomach, spleen, lymph node, bone marrow or kidney. Other embodimentsinclude fluid samples such as bronchial brushes, bronchial washes,bronchial ravages, peripheral blood lymphocytes, lymph fluid, ascites,serous fluid, pleural effusion, sputum, cerebrospinal fluid, lacrimalfluid, esophageal washes, stool or urinary specimens such as bladderwashing and urine.

Bronchial washes sample more area of bronchial epithelium but are alsofrequently cytologically normal. A more complete sampling of therespiratory passages may occur with a bronchiolar alveolar lavage inwhich both left and right proximal and distal small bronchi andbronchioles are washed out.

Nucleic acids are isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementaryDNA.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. The detection may involve indirect identification of theproduct via fluorescent label, chemiluminescence, radioactivescintigraphy of radiolabel or even via a system using electrical orthermal impulse signals (Affymax Technology; Bellus, 1994).Alternatively, the detection may be performed by visual means (e.g.,ethidium bromide staining of a gel).

Following detection, one may compare the results seen in a given samplewith a statistically significant reference group of samples from normalpatients and patients that have or lack alterations in chromosome loci3p21.3 or 10q22. In this way, it is possible to correlate the amount orkind of alterations detected with various clinical states.

B. Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) can be used for molecularstudies. FISH is used to detect highly specific DNA probes which havebeen hybridized to chromosomes using fluorescence microscopy. The DNAprobe is labeled with fluorescent or non fluorescent molecules which arethen detected by fluorescent antibodies. The probes bind to a specificregion or regions on the target chromosome. The chromosomes are thenstained using a contrasting color, and the cells are viewed using afluorescence microscope.

Each FISH probe is specific to one region of a chromosome, and islabeled with fluorescent molecules throughout it's length. Eachmicroscope slide contains many metaphases. Each metaphase consists ofthe complete set of chromosomes, one small segment of which each probewill seek out and bind itself to. The metaphase spread is useful tovisualize specific chromosomes and the exact region to which the probebinds. The first step is to break apart (denature) the double strands ofDNA in both the probe DNA and the chromosome DNA so they can bind toeach other. This is done by heating the DNA in a solution of formamideat a high temperature (70-75° C.) Next, the probe is placed on the slideand the slide is placed in a 37° C. incubator overnight for the probe tohybridize with the target chromosome. Overnight, the probe DNA seeks outit's target sequence on the specific chromosome and binds to it. Thestrands then slowly reanneal. The slide is washed in a salt/detergentsolution to remove any of the probe that did not bind to chromosomes anddifferently colored fluorescent dye is added to the slide to stain allof the chromosomes so that they may then be viewed using a fluorescentlight microscope. Two, or more different probes labeled with differentfluorescent tags can be mixed and used at the same time. The chromosomesare then stained with a third color for contrast. This gives a metaphaseor interphase cell with three or more colors which can be used to detectdifferent chromosomes at the same time, or to provide a control probe incase one of the other target sequences are deleted and a probe cannotbind to the chromosome. This technique allows, for example, thelocalization of genes and also the direct morphological detection ofgenetic defects.

The advantage of using FISH probes over microsatellite instability totest for loss of allelic heterozygosity is that the a) FISH is easilyand rapidly performed on cells of interest and can be used onparaffin-embedded, or fresh or frozen tissue allowing the use ofmicro-dissection b) specific gene changes can be analyzed on a cell bycell basis in relationship to centomeric probes so that truehomozygosity versus heterozygosity of a DNA sequence can be evaluated(use of PCR for microsatellite instability may permit amplification ofsurrounding normal DNA sequences from contamination by normal cells in ahomozygously deleted region imparting a false positive impression thatthe allele of interest is not deleted) c) PCR cannot identifyamplification of genes d) FISH using bacterial artificial chromosomes(BACs) permits easy detection and localization on specific chromosomesof genes of interest which have been isolated using specific primerpairs.

C. Template Dependent Amplification Methods

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety.

Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPO No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention, Walker et al., (1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids, which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). In NASBA, the nucleic acids can be prepared for amplificationby standard phenol/chloroform extraction, heat denaturation of aclinical sample, treatment with lysis buffer and minispin columns forisolation of DNA and RNA or guanidinium chloride extraction of RNA.These amplification techniques involve annealing a primer which hastarget specific sequences. Following polymerization, DNA/RNA hybrids aredigested with RNase H while double stranded DNA molecules are heatdenatured again. In either case the single stranded DNA is made fullydouble stranded by addition of second target specific primer, followedby polymerization. The double-stranded DNA molecules are then multiplytranscribed by an RNA polymerase such as T7 or SP6. In an isothermalcyclic reaction, the RNA's are reverse transcribed into single strandedDNA, which is then converted to double stranded DNA, and thentranscribed once again with an RNA polymerase such as T7 or SP6. Theresulting products, whether truncated or complete, indicate targetspecific sequences.

Davey et al., EPO No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR” (Frohman, M. A., In: PCR PROTOCOLS: A GUIDE TO METHODS ANDAPPLICATIONS, Academic Press, N.Y., 1990; Ohara et al., 1989; eachherein incorporated by reference in their entirety).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu etal., (1989), incorporated herein by reference in its entirety.

D. Southern/Northern Blotting

Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

Briefly, a probe is used to target a DNA or RNA species that has beenimmobilized on a suitable matrix, often a filter of nitrocellulose. Thedifferent species should be spatially separated to facilitate analysis.This often is accomplished by gel electrophoresis of nucleic acidspecies followed by “blotting” on to the filter.

Subsequently, the blotted target is incubated with a probe (usuallylabeled) under conditions that promote denaturation and rehybridization.Because the probe is designed to base pair with the target, the probewill binding a portion of the target sequence under renaturingconditions. Unbound probe is then removed, and detection is accomplishedas described above.

E. Separation Methods

It normally is desirable, at one stage or another, to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography (Freifelder, 1982).

F. Detection Methods

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniquesinvolved are well known to those of skill in the art and can be found inmany standard books on molecular protocols. See Sambrook et al., 1989.For example, chromophore or radiolabel probes or primers identify thetarget during or following amplification.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

In addition, the amplification products described above may be subjectedto sequence analysis to identify specific kinds of variations usingstandard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al, 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences throughout the RPL14, CD39L3, PMGM, or GC20 gene probes thatmay then be analyzed by direct sequencing.

G. Kit Components

All the essential materials and reagents required for detecting andsequencing RPL14, CD39L3, PMGM, or GC20 genes and variants thereof maybe assembled together in a kit. This generally will comprise preselectedprimers and probes. Also included may be enzymes suitable for amplifyingnucleic acids including various polymerases (RT, Taq, Sequenase™ etc.),deoxynucleotides and buffers to provide the necessary reaction mixturefor amplification. Such kits also generally will comprise, in suitablemeans, distinct containers for each individual reagent and enzyme aswell as for each primer or probe.

H. Chip Technologies

Specifically contemplated by the present inventors are chip-based DNAtechnologies such as those described by Hacia et al. (1996) andShoemaker et al. (1996). These techniques involve quantitative methodsfor analyzing large numbers of genes rapidly and accurately. By tagginggenes with oligonucleotides or using fixed probe arrays, one can employchip technology to segregate target molecules as high density arrays andscreen these molecules using methods such as fluorescence, conductance,mass spectrometry, radiolabeling, optical scanning, or electrophoresis.See also Pease et al. (1994); Fodor et al. (1991).

Biologically active DNA probes may be directly or indirectly immobilizedonto a surface to ensure optimal contact and maximum detection. Whenimmobilized onto a substrate, the gene probes are stabilized andtherefore may be used repetitively. In general terms, hybridization isperformed on an immobilized nucleic acid target or a probe molecule isattached to a solid surface such as nitrocellulose, nylon membrane orglass. Numerous other matrix materials may be used, including reinforcednitrocellulose membrane, activated quartz, activated glass,polyvinylidene difluoride (PVDF) membrane, polystyrene substrates,polyacrylamide-based substrate, other polymers such as poly(vinylchloride), poly(methyl methacrylate), poly(dimethyl siloxane),photopolymers (which contain photoreactive species such as nitrenes,carbenes and ketyl radicals capable of forming covalent links withtarget molecules (Saiki, et al., 1994).

Immobilization of the gene probes may be achieved by a variety ofmethods involving either non-covalent or covalent interactions betweenthe immobilized DNA comprising an anchorable moiety and an anchor. DNAis commonly bound to glass by first silanizing the glass surface, thenactivating with carbodimide or glutaraldehyde. Alternative proceduresmay use reagents such as 3-glycidoxypropyltrimethoxysilane (GOP) oraminopropyltrimethoxysilane (APTS) with DNA linked via amino linkersincorporated either at the 3′ or 5′ end of the molecule during DNAsynthesis. Gene probe may be bound directly to membranes usingultraviolet radiation. With nitrocellous membranes, the probes arespotted onto the membranes. A UV light source is used to irradiate thespots and induce cross-linking. An alternative method for cross-linkinginvolves baking the spotted membranes at 80° C. for two hours in vacuum.

Immobilization can consist of the non-covalent coating of a solid phasewith streptavidin or avidin and the subsequent immobilization of abiotinylated polynucleotide (Holmstrom, 1993). Precoating a polystyreneor glass solid phase with poly-L-Lys or poly L-Lys, Phe, followed by thecovalent attachment of either amino- or sulfhydryl-modifiedpolynucleotides using bifunctional crosslinking reagents (Running, 1990and Newton, 1993) can also be used to immobilize the probe onto asurface.

Immobilization may also take place by the direct covalent attachment ofshort, 5′-phosphorylated primers to chemically modified polystyreneplates (“Covalink” plates, Nunc) Rasmussen, (1991). The covalent bondbetween the modified oligonucleotide and the solid phase surface isintroduced by condensation with a water-soluble carbodiimide. Thismethod facilitates a predominantly 5′-attachment of the oligonucleotidesvia their 5′-phosphates.

Nikiforov et al. (U.S. Pat. No. 5,610,287) describes a method ofnon-covalently immobilizing nucleic acid molecules in the presence of asalt or cationic detergent on a hydrophilic polystyrene solid supportcontaining an —OH, —C═O or—COOH hydrophilic group or on a glass solidsupport. The support is contacted with a solution having a pH of about 6to about 8 containing the synthetic nucleic acid and the cationicdetergent or salt. The support containing the immobilized nucleic acidmay be washed with an aqueous solution containing a non-ionic detergentwithout removing the attached molecules.

There are two common variants of chip-based DNA technologies involvingDNA microarrays with known sequence identity. For one, a probe cDNA(500˜5,000 bases long) is immobilized to a solid surface such as glassusing robot spotting and exposed to a set of targets either separatelyor in a mixture. This method, “traditionally” called DNA microarray, iswidely considered as developed at Stanford University. A recent articleby Ekins and Chu (1999) provides some relevant details. The othervariant includes an array of oligonucleotide (20˜25-mer oligos) orpeptide nucleic acid (PNA) probes is synthesized either in situ(on-chip) or by conventional synthesis followed by on-chipimmobilization. The array is exposed to labeled sample DNA, hybridized,and the identity/abundance of complementary sequences are determined.This method, “historically” called DNA chips, was developed atAffymetrix, Inc., which sells its products under the GeneChip®trademark.

VII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

EXAMPLE 1 Lung Cancer Patients and the Correlation between RPL 14 GeneDeletion Percentage and Patient Survival

Tissue Samples Normal lung tissue and cancerous lung tissue wereobtained from lung biopsies embedded in paraffin blocks. These paraffinembedded histologic tissue are from a clinically and pathologically wellcharacterized group of patients with stage 1 lung cancers that underwentresection at M.D. Anderson Cancer Center (MDACC) and were obtained fromMDACC cases on file. These retrospective samples were drawn so that theretrospective samples were as fresh as possible and still have at least2 years follow-up. Many cases had 14 years of follow up. Demographicinformation for these patient groups include: age at the time ofdiagnosis, race, gender, dietary information, initial treatment,screening test with results, date of diagnosis and follow-up withstatus, other diagnosis information, tobacco history, alcohol history,other diagnoses associated with tobacco or alcohol use, and other drugsor treatments which might have a chemopreventative effect.

Cell Dissociation of Interphase Nuclei from Formalin Fixed ParaffinEmbedded Blocks Punch biopsies of 30 histologically representativecancerous lung tissue and adjacent normal lung tissue were performed onparaffin embedded blocks and the resulting tissue sections were placedin 1.5 ml Eppendorf tubes. The same Punch biopsy procedure was performedon 10 controls originating from normal lung tissue with no trace ofcancerous growth. A dewaxing/rehydration incubation protocol, with a 3min centrifuge (12,000 r.p.m.) between each step was performed on thetissue blocks: Xylene (30 min), Xylene (10 min), 100% Ethanol (10 min),95% Ethanol (10 min), 70% Ethanol (10 min), 50% Ethanol (10 min), H₂O(10 min), H₂O (10 min).

After the incubation, scissors were inserted into the 1.5 ml Eppendorftubes and used to finely cut the tissue. This step is critical, as itmechanically removes cells from their connective tissue surroundings.Next, 1 ml Protease K solution was added to each Eppendorf and incubatedat 37° C. for 2 hr, while vortexing every 20 min. After the 37° C.incubation, the tissue contents of the Eppendorfs were poured into nylonmesh covered 15 ml Eppendorf tubes. The 1.5 ml Eppendorf tubes werewashed with PBS and poured again into their respective nylon meshcovered Eppendorfs, in order to minimize sample loss. The nylon meshcovered Eppendorfs were centrifuged at 750 r.p.m.×10 min and thesupernatant was removed with a pipette. Depending on the size of thepellet, between 0.5-2 ml of PBS was added to dilute the cellularspecimen.

Cytospin slides were prepared from the 15 ml Eppendorf tubes. Theconcentration of the 15 ml Eppendorfs were adjusted in accordance withmicroscopic analysis of the cytospin slides. If too many cells werepresent in the field of view, then the Eppendorf tubes were dilutedsubjectively with 0.1 ml aliquots of PBS. If too few were present in thefield of view, then the Eppendorf tubes were re-centrifuged and anestimated amount of supernatant was removed. After appropriateconcentration adjustments, all cytospin slides were placed in a FISHfixative solution (3 parts methanol: 1 part acetic acid) for 20 min.Slides were stored in a −20° C. freezer.

Growth of BAC and Isolation of RPL 14 Template. A colony was inoculatedwith a 10 ml culture containing 1.5 ml LB+ 12.5 μg/ml chloramphenicol.It was grown overnight at 37° C., while shaking at 200 r.p.m. Theculture was transferred to a 1.5 ml mirofuge tube. The cells werepelleted at full speed in a microfuge for 30 sec and the supernatant wasremoved. The cell pellet was thoroughly resuspended in 100 μl chilledSolution I using a Pipetman. 200 μl of freshly prepared Solution II wasadded to the tubes and they were then placed on ice. Each tube was mixed8-10 times via inversion and returned to the ice. The cells lysed andthe solution grew clear and viscous. Next, 150 μl of Solution III wasadded. The tubes were mixed by inversion 8-10 times and returned to theice. The addition of solution III caused the formation of a flocculentprecipitate. The tubes were centrifuged for 6 min at room temperature atfull speed in a microfuge. The supernatant was transferred to a newmicrofuge tube. Any visible debris that was transferred was removed witha toothpick or pipet tip. The DNA was precipitated by adding 1 ml roomtemperature 100% ethanol and centrifuged for 6 min at room temperaturein a microfuge. The supernatant was carefully removed and the DNA pelletwas washed briefly in 70% ethanol. The pellet was air dried briefly(approximately 10 min) before being dissolved in an appropriate volumeof TE buffer.

Preparation of 3p21.3 DNA Probe. The genomic clone of the gene wasisolated from the CITB human BAC (bacterial artificial chromosomes) DNAlibrary pools (Research genetics, Huntsvill, AL0 using PCR techniquewith a specific 3p21.3 gene primer. Genomic DNA was isolated from thisgene by growing the positive BAC clone and isolated gene RPL 14 genomicDNA using a Qiagen Plasmin Kit and following the manufacturesdirections. The gene DNA sequence was confirmed by using PCR with thesame gene primer. Localization of the RPL14 gene on chromosome 3 wasconfirmed by using normal metaphase FISH. Digoxigen is added to theprobed before denaturization of the slides, and follows the procedure inthe Boehringer Mannheim Biochemicals kit. The 3p21.3 template DNAisolated from BAC clones was added to a cocktail of 36.5 ul distilledH₂O, 5 ul A4, 1 ul Digoxigenin-11-dUTP, 1 ul DNA polymerase-1, and 4.0ul of 10× Enzyme mix. The final cocktail was incubated in a 15° C. waterbath for 75 min. The enzymatic reaction was stopped via incubation ofthe cocktail in a 75° C. water bath for 15 min.

The efficiency of these probe depended on its size parameters. Using a100 bp DNA ladder marker and gel electrophoresis, the inventors couldensure that the 3p21.3 probe was between 200-1000 bp size. The markerlane of the gel contained a 10 ul loaded sample: 1 ul 100 bp DNA ladder,2 ul loading buffer, 7 ul 1×TAE. The 3p21.3 lane of the gel contained a10 ul loaded sample: 6 ul 3p21.3 DNA, 2 ul loading buffer, 7 ul 1×TAE.If the banding patterns of the gel showed the p4robe to be between200-100 bp, then the 3p21.3 DAN probe would be ready for precipitation.

Precipitation of 3p21.3 DNA Probe. The probes are precipitated and boundwith a fluorophore using a Nick Translation system (Life Technologies)following the specifications supplied by the manufacturer. 30 ul 3p21.3DNA probe was added to 8.0 ul human Cot-I DNA, 1.0 ul placenta DNA, 3.9ul NaAoc., and 86 ul 100% EtOH. The cocktail was vortexed and brieflycentrifuged. It was stored in a −70° C. freezer for 15 min. Next, it wascentrifuged in a temperature controlled chamber at 4° C. for 20min×136,000 r.p.m. The resulting DNA pellet was air dried for 20 min anddissolved in 60 ul of hybridization buffer. Each slide prepared for FISHanalysis requires 10 ul of hybridization buffer. To the hybridizationbuffer, 4 ul CEP 3 probe (spectrum orange) was added. The final solutionwas denatured in a 75° C. water bath for 5 min and then placed in a 37°C. water bath for 30 min.

FISH Method. Slides were pretreated in a series of 0.;l N HCl-0.2%Triton-X100 in 2×SSC (15 min RT¹, Vibra²) 2×SSC (2 min, RT, Vibra),1×PBS (2 min, RT, Vibra), 1% Formaldehyde (2 min, RT, Vibra), 1%Formaldehyde (4 min RT, No Vibra), 1×PBS ((2 min, RT, Vibra), and 2×SSC((2 min, RT, Vibra). Next, they were denatured in 70% formamide/2×SSC,pH 7.3 for 5 min in a 74° C. water bath. Trial and error showed that atemperature of 74° C. was critical for the production of quality slidesfor FISH. After denaturation, the slides were dehydrated in a coldalcohol series. They wee then subjected to a protease K digestion at 37°C. for 9 min and dehydrated again in a cold alcohol series. After airdrying the slides, 10 ul of the 3p21.3 probe prepared in step 2.5 wasapplied to each slide. They were covered with glass cover slips, sealedwith rubber cement, and allowed to hybridize overnight at 37° C.

Post-washing and Immunohistochemical Labeling. After overnighthybridization, post-hybridization washes occurred in three stages withtwo stages of antibody labeling in between the washes. The first washconsisted of three rinses in 50% Formamide/2×SSC at 45° C. for 10 min.each, two rinses in 2×SSC at 45° C. for 10 min. each, and one rinse2×SSC at room temperature for 10 min. The slides were then blocked with50 ul/slide 4×SSC+1% BSA blocking solution for 5 min. Afterwards, theprimary antibody was diluted in a 1:20 ratio with the blocking solutionand 50 ul was added per slide for 30 min in the dark. The slides werecovered with paraffin cover slips to concentrate the blocking andantibody solutions over the cellular areas. The second wash consisted ofone rinse in 4×SSC at RT for 10 min., one rinse in 4×SSC+1% Triton at RTfor 10 min., one rinse in 4×SSC at RT for 10 min, and one rinse in PN atRT for 10 min. The slides wee then blocked again and labeled with thesecondary antibody, which was prepared in a 1:100 ratio with theblocking solution. The labeling reaction was permitted to occur for 60min. in the dark. The final wash consisted of three rinses in PN at RTfor 10 min. each. Interphase cells wee counterstained with 1 ug/ml DAPIcontaining antifade solution. Ten microliters of DAPI counterstain wereadded to each slide.

Visualization and Scoring of FISH Signals. Hybridization sites wereanalyzed using Nikon microscopes equipped with appropriate filter setsfor visualizing spectrum green and orange as well as DAPI counterstain.At 100 nuclei from each slide were scored using a triple filter. Eachcell was scored individually for the number of RPL 14 signals (spectrumgreen) and the number of corresponding CEP3 (spectrum orange) signals.To avoid misinterpretation due to inefficient hybridization, cells werecounted only if at least one bright CEP3 signal and one bright RPL14signal were present to avoid false monosomies or deletions due toinsufficient hybridization efficiency. Only non-overlapping, intactnuclei were scored. Split centromere signals were counted as one, andminor centromere signals wee disregarded. The inventors used Mantle CellLymphoma cells as a negative control.

TABLE 1 Lung Cancer Patients Deletion Rates of the 3p21.3 probecontaining RPL14, CD39L3, PMGM, and GC20 gene and Patient SurvivalAdeno- carcinoma Deletion Squamous Deletion Cases Percent Expired CasesPercent Expired 1a 3% dead 8a 4% dead 1b 10% 8b 44% 2a 2% alive 9a 8%alive 2b 11% 9b 64% 3a 8% alive 10a 6% alive 3b 16% 10b 8% 4  50% dead11  58% dead 5  2% dead 12a 6% alive 6a 14% alive 12b 10% 6b 30% 7 44%dead

Discussion. Table 1 provides an organized view of 12 patients sufferingfrom lung cancer. The patients were separated into two different groups:those with adenocarcinoma and those with squamous cell carcinoma. Forexample, 3b represents cells isolated from a bronchous tumor via punchbiopsies of paraffin embedded tissue blocks. The partner number 3arepresents cells isolated from the same paraffin block, but from anontumorous bronchous. Next, two types of cell samples were isolated perpatient. Using FISH techniques directed to the Centromere 3 and theRPL14 gene of dissociated cells, the inventors were able to determinethe deletion rate of the RPL14 gene in all patients. Initial data showsa promising correlation between the deletion percentage and survival ofa patient.

EXAMPLE 2 Retrospective Study of Lung Cancer Using 3p21.3 gene probe andFISH detection

From an initial population of 200 patients studied retrospectively withStage I lung cancer (culled from >13,000 patient files 1987-1988) theinventors identified 100 patients who had relapsed or died within 5years. Additionally the inventors obtained archival bronchial tissuefrom 100 patients with lung tissue removed for reasons other than cancerwhich formed the basis of the control group. A detailed demographichistory including smoking status, occupational history and familyhistory of cancer was obtained for each patient.

The RPL14 gene probe (located on 3p21.3). Specific primer was designedbased on the gene sequence with Electronic-PCR software,. The genomicclone of the gene was isolated from the CITB human BAC DNA library pools(Research genetics, Huntsville, Ala.) using PCR technique with thisspecific gene primer.

Isolation of genomic DNA of 3p21,3. Growth of the positive BAC clone andisolated gene RPL14 genomic DNA using Qiagen Plasmid Kit as instructedby the manufacture. The gene DNA sequence was confirmed by using PCRwith the same gene primer. Localization of the RPL14 gene on chromosome3 was confirmed by using normal metaphase FISH. Preparation of specificgene FISH probes were prepared using a Nick Translation System (LifeTechnologies) as instructed by the manufacturer. If the banding patternsof the gel showed the probe to be between 200-10000 bp, then the probewould be ready for precipitation.

The BAC clone that contained genomic sequences that have the highestfrequency of deletion at 10q region in tumor cell lines was selected.The DNA of this BAC clone isolation and probes labeling procedure wereperformed as above.

Tissue samples. Punch biopsies of histologically representativecancerous lung tissue and adjacent normal or histologically abnormalbronchial epithelial tissues were performed on paraffin embedded blocks.Resulting tissue sections were digested to obtain cell dissociation ofinterphase nuclei according to the Hedley technique. A subset of caseshad imprints obtained form tumor and adjacent bronchus. Cells were fixedin FISH fixative which is Carnoy's solution (methanol and acetic acid ina 3:1 ratio).

FISH Studies. Dual color FISH studies were performed with the SpectrumOrange centromeric probes for chromosome 3 (CEP 3) (Vysis) anddigoxigenin labeled specific RLP 14 gene or the Spectrum Orangecentromeric probes for chromosome 10 (CEP 10) (Vysis) and Digoxigeninlabeled specific 10q22 probes. CEP 3 or 10 probes were used as controlprobes respectively.

Slides were denatured in 70% formamide at 73° C. for 5 min. A mix ofprobes were denatured for 5 min at 75° C. and then applied to slides.After overnight hybridization at 37° C., post-hybridization washing wereas follows: 50% Formamdie/2×SSC at 45° C. for 5 min. Digoxigenin labeledspecific gene or 10q22 probes are detected by FITC conjugated sheepantidigoxigenin.

Interphases were counterstained with DAPI or PI counterstaining antifadesolution. Hybridization sites were analyzed using Nikon microscopesequipped with the appropriate filter sets for visualizing spectrum greenor orange as well as nuclei Counterstain. At least 200 nuclei withsignals from each probe were scored using a triple filter. Slides wereanalyzed only if 80% of the cells were interpretable in the field ofview. Only non-overlapping, intact nuclei were scored. Split centromericsignals (distance between two signals was equal or less than 0.5 um)were counted as one, and minor centromeric signals were disregarded.Normal lymphocytes were used as external control Deletion (%) wasdefined percent of cells with fewer signals of specific probe thansignals of CEP3 or CEP10 in 200 cells counted.

Results. To date, the inventors have examined numerous dissociatedtumors with their adjacent bronchi as well as numerous controls. Basedon the results using DNA probes from 3p and 10q, the inventors haveshown that the probes most likely are detecting tumor suppressor genesthat are lost early on in tumorigenesis, are associated with smoking andappear to predict for the development of non-small cell lung cancer aswell as for its overall survival. In addition, the inventors have shownthat non-smokers who develop lung cancer have much higher rates ofdeletions, higher even than smokers (FIGS. 1-2) and that these resultsare statistically significant (p<0.001).

FIG. 1 and FIG. 2 show the 3p21 and 10q22 deletion rates in adjacentbronchial epithelial cells of patients with benign lung disease,patients who developed stage 1 non-small cell cancer that did notrelapse, and patients with stage 1 non-small cell cancer with relapse.Note that patients who relapsed had a much higher level of deletionsthan those patients who did not relapse, regardless of smoking status.

3p and 10q deletions were frequently expressed in lung tumors and showedno correlation with relapse, however the presence of 3p and 10qabnormalities in adjacent bronchial tissue was strongly correlated withrelapse (0.09, and 0.0279) and survival (p=0.0348). Therefore, theprobes may be useful markers in smoking-related damaged epithelium forrisk assessment and for monitoring the efficiency of chemopreventiveregimes.

EXAMPLE 3 Lung Cancer Susceptibility in Former Heavy Smokers

A subset of bronchial lavages from former heavy smokers who had quit foran average of 6 years with median pack year history of 46 years wasstudied for lung cancer susceptibility. The study patients havesurveillance bronchoscopy followed by blind biopsies of main bronchifrom both lungs. Following this, a bronchial wash was performed, andtriaged. Even though all these patients had quit smoking (average 6years previously) most showed significant deletions for the 3p21 or the10q22 FISH probes in bronchial wash specimens, indicating that ingenetically susceptible individuals molecular defects appear to bepersistent and are not related to the number of pack years.

EXAMPLE 4 Serial Dilution of Tumor Cells

To detect how low concentration of tumor cells in the bronchial washingsample can be detected and the actual number and distribution of thegene in individual morphological cells, a serial dilution was done forevaluating the sensitivity of the 3p21.33 FISH probe. This test was alsofor quality control purpose. Two cell lines were used for the serialdilution experiment. H-1792 lung adenocarcinoma cell line was obtainedfrom ATCC, the cell line exhibited cytogentic abnormalities includingtrisome chromosome 3 and 3p21.33 deletion. By FISH analysis, the celldemonstrated that over 100% of the interphases had 3 signals of CEP3 incontrast to 2 signals of 3p21.33 with 3p21.33/CEP11 probes. The normalbronchial epithelial cell was derived from a normal individual, showingnormal number and structure of chromosome 3. H-1792 cells were mixedwith same number of normal epithelial to dilute H-1792 cells to 50%. Aserials of dilution was performed to further dilute H-1792 cell to 25%,12.5%, 6.3%, 3.1%, 1.6%, 0.8%. The slides were made by cytospinpreparations and randomized before hybridization. After hybridizationand post washing, the percentage of cells with deletion of 3p12.33signals were counted and compared with the projected values, as shown inFIG. 4.

Results of the serial dilution experiment demonstrated that the dilutionconcentration was positively related to the percentage of 3p21.33deletion cells detected by FISH. However, when tumor cell line werediluted to a concentration ≦3.1%, it was not possible to identify 3p21.33 deletion cells, suggesting that the sensitivity of the probe or thelowest concentration of positive cells detected by the probe inbronchial washings was 3.1%.

EXAMPLE 5 Progression of GC20 Study

After narrowing down the critical gene region in 3p21, the novel geneSUI1/GC20 (SEQ ID NO: 7) was identified in the region. SUI1/GC20 is ahomolog of the SUI1 gene, which is a superfamily consisting of a growingnumber of proteins; SUI1 is a 113 amino-acid polypeptide similar to theprotein from various different species. Primarily, the SUI1 gene productwas believed to be a monitor translational accuracy protein byrecognition of the protein synthesis initiation codon. Recent studiesdemonstrated that the SUI1 protein has a role in the nonsense-mediatedmRNA decay pathway, by which cells have evolved elaborate mechanisms torid themselves of aberrant proteins and transcripts. Identification of astress-inducible cDNA of SUI1 suggested that modulation of translationinitiation occurs during cellular stress and may represent an importantadaptive response to genotoxin (e.g., tobacco) as well as endoplasmicreticular stress. SUI1 was expressed in normal liver but not in livercarcinoma cells. Introduction of SUI1 into liver carcinoma cellsinhibited cell growth in vitro and partially inhibited tumor formationin nude mice. It is rational to suggest, therefore, proteins of the SUI1family possess tumor-suppressing properties and may represent a primaryevent, rather than a consequence, of tumorigenesis. Furthermore, sincedeletion of 3p21.3 was found by others to be the earliest acquiredgenetic changes in the pathogenesis of lung cancer, inventors also foundthat SUL1/GC20 transcript was diminished in all lung cancer cell linestested by reverse transcription-PCR (RT-PCR). Inventors have cloned thefull-length cDNA of SUI1/GC20 into a constitutive (pcDNA3.1/GS) with theC-terminal V5 epitope and polyhistidine (6xHis) tag (SEQ ID NO: 9). Thefirst four nucleic acids of SEQ ID NO: 9, “cacc” were added to theinsert before the ATG by including the sequence in the forward primer(SEQ ID NO: 11), in order to conform to the to the consensus Kozaksequence for optimal translation initiation. The reverse primer is givenin SEQ ID NO: 10 The last 102 nucleic acids of SEQ ID NO: 9 is not partof the insert, but is derived from the vector pCDNA3.1/GS, and codes forthe V5/6x His tag.

The non-small cell lung cancer cell line H1972 was transfected with thefull-length cDNA of SUI1/GC20 (SEQ ID NO: 9), resulting in H-1972pcDNA3.1/GC20 or with the vector pcDNA3.1/GS (resulting in H-1972pcDNA3.1) as control. Protein expression of GC20 was detected in theH-1972 pcDNA3.1/GC20 cells, but not in control cells. The growth ofH-1972 pcDNA3.1/GC20 cells in serum-containing medium was significantlyslower than that of H-1972 or the vector-transfected control H-1972pcDNA3.1.

Studying the molecular genetic mechanisms by which SUI1/GC20 isinactivated ca be done to characterize the gene. Analyze of the functionof the gene can also be used to demonstrate its ability to inhibit cellgrowth and suppress tumorgenicity. As results of these studies, there isa better understanding of the tumorigenesis of tobacco-related lungcancer and the clinical biomarkers useful for its early detection andrisk assessment.

EXAMPLE 6 FISH Studies on Bronchial Wash Specimens from Patients withBenign, Atypical and Malignant Cytology Using a 3p21.3 DNA Probe

Bronchial wash specimens were tested for deletions of 3p21.3 by FISHusing the locus-specific probe for 3p21.3 together with a centromericprobe for chromosome 3 as control. Inventors tested patients withnon-small cell bronchogenic carcinoma, and patients on a chemopreventiveprotocol who demonstrated by cytology metaplasia, reserve cellhyperplasia or no abnormality. Also, the presence of the 3p deletion wascorrelated with the number of pack-years of smoking or tobacco-use.

Negative Cytology 7 cases with negative cytology showed levels ofdeletions of 3p21.3 between 0%-13% (mean 7.2±0.05) when tested with the3p21.3 probe. The highest level of deletion was associated with a 122.5pack year history of smoking. Interestingly, this high level of deletionwas noted in two specimens, 6 months apart, from the same patient,indicating that there is a consistent deletion that did not response tofenretinamide (or cis-retinoic acid) therapy that was used as achemopreventive agent.

Atypical Cytology There were 6 cases with cytological evidence of eitherreserve cell hyperplasia or squamous metaplasia/atypical metaplasia. Thehighest level of deletion was noted in a 90-pack year smoker. Thedeletions ranged from 7% to 15% (mean=10.5%±0.036).

Carcinoma In the third category of patients with cytological evidence ofcarcinoma (2 squamous carcinoma, 1 adenocarcinoma), the mean percentdeletion was 17±0.13 (range: 8%-23%).

Results These results showed that in patients without evidence of lungcancer/squamous atypia, who had a history of smoking, a deletion of3p21.3 existed that roughly paralleled the number of pack years smokedindicating that this deletion may occur secondarily to exposure totobacco smoke, and also may be an early event in neoplastictransformation. None of these patients have yet to evidence clinical orstraight chest X-ray evidence of lung cancer, however, those with thehighest levels of deletion may be at high risk to develop neoplasia.

In patients with atypia as manifested by squamous metaplasia or atypia,the level of deletion was higher than in the negative group, with thehighest levels of deletion noted in patients with carcinoma.

The results also correspond with previous studies with 3p21.3 probe forchromosomal aberrations in microdissected lung carcinomas and adjacent“normal” bronchial cells. Genetic instability is a very early event intumorigenesis and chromosomal numerical abnormalities are associatedwith smoking. 3p21.3 deletions occurred more frequently in the lungtumors and adjacent bronchi of the patients who smoked than in controllung tissue from patients who did not smoke. Smoking may cause moleculardamage much earlier than the corresponding manifestation of neoplasia ata morphologic level. Smoking is a major etiologic factor for thedevelopment of lung cancer and based on the studies presented herein,the loss of 3p21.3 is an early event in the tumorigenesis of lungcancer.

The 3p21.3 probe will be a useful marker in monitoring smoking-relatedtarget epithelia to measure risk assessment and for monitoring theefficiency of chemo-prevention therapy in high-risk former or currentsmokers.

TABLE 2 Results of FISH Studies on Bronchial Wash Specimens fromPatients with Benign, Atypical and Malignant Cytology Using a 3p21.3 DNAProbe. DATE SMOKE HX BW # MDA # Received (Pack Years) DIAGNOSIS 3pFISHBenign 87 228891 5/11/00 67.5 No slide 0% 179 398860 10/12/00 122.5Negative 12% 212 398860 1/12/01 122.5 Negative 13% 247 451844 5/1/01 45Negative 12% 249 424531 5/4/01 44 Negative 5% 257 459858 6/26/01 39Negative 8% 258 458362 7/12/01 26 Negative 1% Atypia 31 413570 1/19/0087.5 Metaplasia 12% 146 385669 9/1/00 Non-Smoker Metaplasia 8% 243475347 4/26/01 38 Metaplasia 14% 244 474853 4/26/01 30 Metaplasia 7% 246475666 4/27/01 90 Metaplasia 15% 256 429515 6/8/01 75 reserve cell 7%hyperplasia Malignant 252 404860 5/8/01 60 Sq. CA 11% 127 358000 8/3/00Non-Smoker Sq. CA 8% 210 406098 1/10/01 Non-Smoker Ad. CA 32

EXAMPLE 7 Sensitivity of the 3p21.33 FISH Probe in Detecting Lung CancerCells in Bronchial Wash Specimens

It was hypothesized that deletions of 3p.21.33 may be detected early onin carcinogenesis, and may thus have the potential to predict apatient's predisposition towards developing either primary lung canceror a relapse thereof. The purpose of this study was to explore theefficacy of the FISH test, specifically the sensitivity of the 3p21.33probe, for determining 3p21.33 deletions in interphase cells frompatients' bronchial samples with the aim of developing a method fordetermining genetic predisposition to lung cancer. The sensitivity ofthe outcome depended on the visibility of the 3p21.33 gene loci as wellas the ability to detect deletions of the 3p21.33 locus in the malignantcell lines compared to the admixed normal bronchial cells.

Cell Samples and Slides The tumor cells in this study were obtained froman H-1792 lung adenocarcinoma cell line obtained from ATCC, whichexhibited cytogenic abnormalities including trisomy of chromosome 3 and3p21.33 deletion. These cells were separated and harvested from culturebottles and diluted from a concentration of 2.52×10⁶ cells per ml to7.14×10⁵ cells per ml using PBS buffer. Normal bronchial epithelialcells, showing normal numbers and structure for chromosome 3, wereacquired from the bronchial wash of a normal individual at aconcentration of 7.14×10⁵ cells per ml. The cancer cell sample wasdiluted by the normal cells to concentrations of 0%, 0.8%, 1.6%, 3.13%,6.25%, 12.5%, 25%, 50%, 75%, 87.5%, 93.75%, 96.8%, 98.4%, and 100% andtransferred onto individual slides using a cytospin preparation.

Nick Translation The DNA probe used to identify the 3p21.33 gene wascreated using nick translation, a widely used method for its ease incontrolling fragment size (Wilkinson, 1998) Digoxigenin enzyme, which isable to be detected with antibodies, was used to cut 3p DNA (Andreeff etal., 1999). The probe was tested using gel electrophoresis to ensure alength of 200-500 kilobase pairs. CEP3 probe (chromosome 3 centromere)was premixed and provided by a commercial company (Vysis, Downers Grove,Ill.).

FISH Method The 3p21.33 probe was precipitated by mixing with humancot-1 DNA (Vysis, Downers Grove, Ill.), human placenta DNA (Sigma, LaJolla, Calif.), NaOAcetate, and 100%-20 ethanol, incubated at −80° C.for 15 minutes, and centrifuged for 20 minutes at 4° C. The remainingpellet was dissolved in hybridization buffer (Vysis, Downers Grove,Ill.) at room temperature, at 10 μl per slide, and the centromeric probewas added. The probe was placed in a 75° C. water bath for 5 minutes andthen transferred to a 37° C. water bath for 20 minutes. At the sametime, the slides to be tested were placed into a 70% Formamide/SSCsolution for 3 to 4 minutes to denature the DNA and subsequently placedinto a series of cold ethanol jars in order to permeabilize the cells byremoving the lipid membranes (Wilkinson, 1998). 10 μl of the probe wasthen pipetted onto each slide and put into a humidity box to incubateovernight at 37° C. The following day, the slides were first placed into3 jars of 50% Formamide/2×SSC at 45° C. for 10 minutes each and theninto a jar of 2×SSC for 10 minutes at 45° C. as well. The slides wereblocked for five minutes using 4×SSC/BSA and then covered with the firstantibody (anti-digoxigenin) and placed in a humidity box for an hour.This was followed by a series of TNT buffer washings. Once again theblocking procedure was performed before the second antibody was placedon the slides for another hour. The slides were washed again with TNTbuffer. Lastly, 10 μl of DAPI was added to the slides to stain thenucleus of each cell.

Visualization and Counting of FISH Signals The hybridized slides wereexamined utilizing a Labophot-2 microscope (Nikon, Tokyo, Japan) underfilters for visualizing green, orange, and DAPI fluorescence signals(FIGS. 5-9). One hundred cells were selected for analysis from eachdilution, and were counted only if the entire nucleus was distinct andintact. To avoid misinterpretations owing to false monosomies ordeletions due to insufficient hybridization, nuclei were counted only ifat least one bright CEP3 signal (orange) and one bright 3p21.33 signal(green) were present. The numbers of orange signals versus green signalswere counted and recorded individually for each chosen cell. Aparticular cell was deemed a normal epithelial cell if it had an equalnumber of green and orange signals, signifying that there was one3p.21.33 gene per centromere on the chromosomes. In contrast, cells wereidentified as tumor cells if they possessed fewer green signals thanorange signals, proving that there, indeed, was a deletion of the3p21.33 gene within its genomic makeup.

Results and Discussion Typically, normal epithelial cells displayed 2orange and 2 green signals, whereas tumor cells showed 3 orange and 2green signals (FIGS. 5-9). Data indicates that the baseline sensitivityfor FISH detection of deletions of 3p21.33 is 3.13% (see Example 4),thisplaces high hopes for early detection of the development lungcancer, recurrence, or metastasis. With extended studies, the FISH probefor 3p21.33 can be established and widely used as a useful and reliablemarker for assessing lung cancer and, perhaps, further utilizing andimproving methods like fine needle aspiration in conjunction with theFISH method. This study also lays the baseline for future studies andfor the monitoring of preneoplastic or neoplastic events and may be usedas a surrogate intermediate biomarker in chemoprevention techniques inlung cancer.

EXAMPLE 8 Statistical Analysis of 10Q Probe

FIG. 10 and FIG. 11 provide a predicted probability of relapse and longterm survival for patients. Using data from 96 patients, the deletion of10Q in bronchial epithelial cells adjacent to the tumor cells iscompared with both relapse (FIG. 10 and long term survival (FIG. 11).10Q deletion is a significant predictor of relapse.

In FIG. 12, the proportion of patients who are relapse free at timesranging from 0 to 108 months is shown. The data is divided into a set ofpatients who have a N10q value of greater than 5 and patients who have aN10q value of less than or equal to 5. While about 40% of the patientswith N10q>5 are relapse free after a long interval (5-7 years), over 60%of the patients with N10q≦5 are relapse free after the same timeinterval.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

Wilkinson, D. G., In Situ Hybridization: A Practical Approach. New York,Oxford: 1998.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 11 <210> SEQ ID NO 1 <211> LENGTH: 722<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (18)..(680) <400> SEQUENCE: 1cgcctaacgc tgccaac atg gtg ttc agg cgc ttc gtg #gag gtt ggc cgg        50                   # Met Val Phe Arg Arg Phe Val Glu Val Gly  #Arg                   #   1                #5                   #10gtg gcc tat gtc tcc ttt gga cct cat gcc gg#a aaa ttg gtc gcg att       98Val Ala Tyr Val Ser Phe Gly Pro His Ala Gl #y Lys Leu Val Ala Ile             15      #             20      #             25gta gat gtt att gat cag aac agg gct ttg gt#c gat gga cct tgc act      146Val Asp Val Ile Asp Gln Asn Arg Ala Leu Va #l Asp Gly Pro Cys Thr         30          #         35          #         40caa gtg agg aga cag gcc atg cct ttc aag tg#c atg cag ctc act gat      194Gln Val Arg Arg Gln Ala Met Pro Phe Lys Cy #s Met Gln Leu Thr Asp     45              #     50              #     55ttc atc ctc aag ttt ctg cac agt gcc cac ca#g aag tat gtc cga caa      242Phe Ile Leu Lys Phe Leu His Ser Ala His Gl #n Lys Tyr Val Arg Gln 60                  # 65                  # 70                  # 75gcc tgg cag aag gca gac atc aat aca aaa tg#g gca gcc aca cga tgg      290Ala Trp Gln Lys Ala Asp Ile Asn Thr Lys Tr #p Ala Ala Thr Arg Trp                 80  #                 85  #                 90gcc aag aag att gaa gcc aga gaa agg aaa gc#c aag atg aca gat ttt      338Ala Lys Lys Ile Glu Ala Arg Glu Arg Lys Al #a Lys Met Thr Asp Phe             95      #            100      #            105gat cgt ttt aaa gtt atg aag gca aag aaa at#g agg aac aga ata atc      386Asp Arg Phe Lys Val Met Lys Ala Lys Lys Me #t Arg Asn Arg Ile Ile        110           #       115           #       120aag aat gaa gtt aag aag ctt caa aag gca gc#t ctc ctg aaa gct tct      434Lys Asn Glu Val Lys Lys Leu Gln Lys Ala Al #a Leu Leu Lys Ala Ser    125               #   130               #   135ccc aaa aaa gca cct ggt act aag ggt act gc#t gct gct gct gct gct      482Pro Lys Lys Ala Pro Gly Thr Lys Gly Thr Al #a Ala Ala Ala Ala Ala140                 1 #45                 1 #50                 1 #55gct gct gct gct gct gct gct gct gct aaa gt#t cca gca aaa aag atc      530Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Va #l Pro Ala Lys Lys Ile                160   #               165   #               170acc gcc gcg agt aaa aag gct cca gcc cag aa#g gtt cct gcc cag aaa      578Thr Ala Ala Ser Lys Lys Ala Pro Ala Gln Ly #s Val Pro Ala Gln Lys            175       #           180       #           185gcc aca ggc cag aaa gca gcg cct gct cca aa#a gct cag aag ggt caa      626Ala Thr Gly Gln Lys Ala Ala Pro Ala Pro Ly #s Ala Gln Lys Gly Gln        190           #       195           #       200aaa gct cca gcc cag aaa gca cct gct cca aa#g gca tct ggc aag aaa      674Lys Ala Pro Ala Gln Lys Ala Pro Ala Pro Ly #s Ala Ser Gly Lys Lys    205               #   210               #   215gca taa gtggcaatca taaaaagtaa taaaggttct ttttgacctg tt#               722 Ala 220 <210> SEQ ID NO 2 <211> LENGTH: 220<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Met Val Phe Arg Arg Phe Val Glu Val Gly Ar #g Val Ala Tyr Val Ser  1               5  #                 10  #                 15Phe Gly Pro His Ala Gly Lys Leu Val Ala Il #e Val Asp Val Ile Asp             20      #             25      #             30Gln Asn Arg Ala Leu Val Asp Gly Pro Cys Th #r Gln Val Arg Arg Gln         35          #         40          #         45Ala Met Pro Phe Lys Cys Met Gln Leu Thr As #p Phe Ile Leu Lys Phe     50              #     55              #     60Leu His Ser Ala His Gln Lys Tyr Val Arg Gl #n Ala Trp Gln Lys Ala 65                  # 70                  # 75                  # 80Asp Ile Asn Thr Lys Trp Ala Ala Thr Arg Tr #p Ala Lys Lys Ile Glu                 85  #                 90  #                 95Ala Arg Glu Arg Lys Ala Lys Met Thr Asp Ph #e Asp Arg Phe Lys Val            100       #           105       #           110Met Lys Ala Lys Lys Met Arg Asn Arg Ile Il #e Lys Asn Glu Val Lys        115           #       120           #       125Lys Leu Gln Lys Ala Ala Leu Leu Lys Ala Se #r Pro Lys Lys Ala Pro    130               #   135               #   140Gly Thr Lys Gly Thr Ala Ala Ala Ala Ala Al #a Ala Ala Ala Ala Ala145                 1 #50                 1 #55                 1 #60Ala Ala Ala Ala Lys Val Pro Ala Lys Lys Il #e Thr Ala Ala Ser Lys                165   #               170   #               175Lys Ala Pro Ala Gln Lys Val Pro Ala Gln Ly #s Ala Thr Gly Gln Lys            180       #           185       #           190Ala Ala Pro Ala Pro Lys Ala Gln Lys Gly Gl #n Lys Ala Pro Ala Gln        195           #       200           #       205Lys Ala Pro Ala Pro Lys Ala Ser Gly Lys Ly #s Ala     210              #   215               #   220 <210> SEQ ID NO 3 <211> LENGTH: 2797<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (83)..(1672) <400> SEQUENCE: 3acccacgcgt ctggccgcgg gccgcctctg cggcagcgct agtcgccttc tc#cgaatcgg     60 ctccgcacag ctaggagaaa ag atg ttc act gtg ctg acc# cgc caa cca tgt     112                   #       Met Phe Thr Val Leu Thr A #rg Gln Pro Cys                   #         1          #      5             #      10gag caa gca ggc ctc aag gcc ctc tac cga ac#t cca acc atc att gcc      160Glu Gln Ala Gly Leu Lys Ala Leu Tyr Arg Th #r Pro Thr Ile Ile Ala                 15  #                 20  #                 25ttg gtg gtc ttg ctt gtg agt att gtg gta ct#t gtg agt atc act gtc      208Leu Val Val Leu Leu Val Ser Ile Val Val Le #u Val Ser Ile Thr Val             30      #             35      #             40atc cag atc cac aag caa gag gtc ctc cct cc#a gga ctg aag tat ggt      256Ile Gln Ile His Lys Gln Glu Val Leu Pro Pr #o Gly Leu Lys Tyr Gly         45          #         50          #         55att gtg ctg gat gcc ggg tct tca aga acc ac#a gtc tac gtg tat caa      304Ile Val Leu Asp Ala Gly Ser Ser Arg Thr Th #r Val Tyr Val Tyr Gln     60              #     65              #     70tgg cca gca gaa aaa gag aat aat acc gga gt#g gtc agt caa acc ttc      352Trp Pro Ala Glu Lys Glu Asn Asn Thr Gly Va #l Val Ser Gln Thr Phe 75                  # 80                  # 85                  # 90aaa tgt agt gtg aaa ggc tct gga atc tcc ag#c tat gga aat aac ccc      400Lys Cys Ser Val Lys Gly Ser Gly Ile Ser Se #r Tyr Gly Asn Asn Pro                 95  #                100  #                105caa gat gtc ccc aga gcc ttt gag gag tgt at#g caa aaa gtc aag ggg      448Gln Asp Val Pro Arg Ala Phe Glu Glu Cys Me #t Gln Lys Val Lys Gly            110       #           115       #           120cag gtt cca tcc cac ctc cac gga tcc acc cc#c att cac ctg gga gcc      496Gln Val Pro Ser His Leu His Gly Ser Thr Pr #o Ile His Leu Gly Ala        125           #       130           #       135acg gct ggg atg cgc ttg ctg agg ttg caa aa#t gaa aca gca gct aat      544Thr Ala Gly Met Arg Leu Leu Arg Leu Gln As #n Glu Thr Ala Ala Asn    140               #   145               #   150gaa gtc ctt gaa agc atc caa agc tac ttc aa#g tcc cag ccc ttt gac      592Glu Val Leu Glu Ser Ile Gln Ser Tyr Phe Ly #s Ser Gln Pro Phe Asp155                 1 #60                 1 #65                 1 #70ttt agg ggt gct caa atc att tct ggg caa ga#a gaa ggg gta tat gga      640Phe Arg Gly Ala Gln Ile Ile Ser Gly Gln Gl #u Glu Gly Val Tyr Gly                175   #               180   #               185tgg att aca gcc aac tat tta atg gga aat tt#c ctg gag aag aac ctg      688Trp Ile Thr Ala Asn Tyr Leu Met Gly Asn Ph #e Leu Glu Lys Asn Leu            190       #           195       #           200tgg cac atg tgg gtg cac ccg cat gga gtg ga#a acc acg ggt gcc ctg      736Trp His Met Trp Val His Pro His Gly Val Gl #u Thr Thr Gly Ala Leu        205           #       210           #       215gac tta ggt ggt gcc tcc acc caa ata tcc tt#c gtg gca gga gag aag      784Asp Leu Gly Gly Ala Ser Thr Gln Ile Ser Ph #e Val Ala Gly Glu Lys    220               #   225               #   230atg gat ctg aac acc agc gac atc atg cag gt#g tcc ctg tat ggc tac      832Met Asp Leu Asn Thr Ser Asp Ile Met Gln Va #l Ser Leu Tyr Gly Tyr235                 2 #40                 2 #45                 2 #50gta tac acg ctc tac aca cac agc ttc cag tg#c tat ggc cgg aat gag      880Val Tyr Thr Leu Tyr Thr His Ser Phe Gln Cy #s Tyr Gly Arg Asn Glu                255   #               260   #               265gct gag aag aag ttt ctg gca atg ctc ctg ca#g aat tct cct acc aaa      928Ala Glu Lys Lys Phe Leu Ala Met Leu Leu Gl #n Asn Ser Pro Thr Lys            270       #           275       #           280aac cat ctc acc aat ccc tgt tac cct cgg ga#t tat agc atc agc ttc      976Asn His Leu Thr Asn Pro Cys Tyr Pro Arg As #p Tyr Ser Ile Ser Phe        285           #       290           #       295acc atg ggc cat gta ttt gat agc ctg tgc ac#t gtg gac cag agg cca      1024Thr Met Gly His Val Phe Asp Ser Leu Cys Th #r Val Asp Gln Arg Pro    300               #   305               #   310gaa agt tat aac ccc aat gat gtc atc act tt#t gaa gga act ggg gac      1072Glu Ser Tyr Asn Pro Asn Asp Val Ile Thr Ph #e Glu Gly Thr Gly Asp315                 3 #20                 3 #25                 3 #30cca tct ctg tgt aag gag aag gtg gct tcc at#a ttt gac ttc aaa gct      1120Pro Ser Leu Cys Lys Glu Lys Val Ala Ser Il #e Phe Asp Phe Lys Ala                335   #               340   #               345tgc cat gat caa gaa acc tgt tct ttt gat gg#g gtt tat cag cca aag      1168Cys His Asp Gln Glu Thr Cys Ser Phe Asp Gl #y Val Tyr Gln Pro Lys            350       #           355       #           360att aaa ggg cca ttt gtg gct ttt gca gga tt#c tac tac aca gcc agt      1216Ile Lys Gly Pro Phe Val Ala Phe Ala Gly Ph #e Tyr Tyr Thr Ala Ser        365           #       370           #       375gct tta aat ctt tca ggt agc ttt tcc ctg ga#c acc ttc aac tcc agc      1264Ala Leu Asn Leu Ser Gly Ser Phe Ser Leu As #p Thr Phe Asn Ser Ser    380               #   385               #   390acc tgg aat ttc tgc tca cag aat tgg agt ca#g ctc cca ctg ctg ctc      1312Thr Trp Asn Phe Cys Ser Gln Asn Trp Ser Gl #n Leu Pro Leu Leu Leu395                 4 #00                 4 #05                 4 #10ccc aaa ttt gat gag gta tat gcc cgc tct ta#c tgc ttc tca gcc aac      1360Pro Lys Phe Asp Glu Val Tyr Ala Arg Ser Ty #r Cys Phe Ser Ala Asn                415   #               420   #               425tac atc tac cac ttg ttt gtg aac ggt tac aa#a ttc aca gag gag act      1408Tyr Ile Tyr His Leu Phe Val Asn Gly Tyr Ly #s Phe Thr Glu Glu Thr            430       #           435       #           440tgg ccc caa ata cac ttt gaa aaa gaa gtg gg#g aat agc agc ata gcc      1456Trp Pro Gln Ile His Phe Glu Lys Glu Val Gl #y Asn Ser Ser Ile Ala        445           #       450           #       455tgg tct ctt ggc tac atg ctc agc ctg acc aa#c cag atc cca gct gaa      1504Trp Ser Leu Gly Tyr Met Leu Ser Leu Thr As #n Gln Ile Pro Ala Glu    460               #   465               #   470agc cct ctg atc cgt ctg ccc ata gaa cca cc#t gtc ttt gtg ggc acc      1552Ser Pro Leu Ile Arg Leu Pro Ile Glu Pro Pr #o Val Phe Val Gly Thr475                 4 #80                 4 #85                 4 #90ctc gct ttc ttc aca gtg gca gcc ttg ctg tg#t ctg gca ttt ctt gca      1600Leu Ala Phe Phe Thr Val Ala Ala Leu Leu Cy #s Leu Ala Phe Leu Ala                495   #               500   #               505tac ctg tgt tca gca acc aga aga aag agg ca#c tcc gag cat gcc ttt      1648Tyr Leu Cys Ser Ala Thr Arg Arg Lys Arg Hi #s Ser Glu His Ala Phe            510       #           515       #           520gac cat gca gtg gat tct gac tga gccttcaaag ca#gctcctgg agtccaatgg     1702 Asp His Ala Val Asp Ser Asp        525           #       530ctgcttagag tcagcctggg tggcaccagg caatgcaggt gaagtggctg cc#ttcaggaa    1762atacaactaa ctaaaatcaa acacctaggt cacgtgcctc tcaaatactg at#ttctgcca    1822cagcacctct tgaggcatcc cttggctatt ctgtgcatat tgttcttcag ag#acctcact    1882acccacatgc tgatctattg gggaacagag aagagacagg ccactaaggt ca#ggctcttt    1942atattaagtt ccccagagga agagtaagtt gagaaggtat cagtttaatg tt#gaagaatt    2002gacctcaggg ctcagtttcc atttccctcc ctcagtattc ttcctggcaa ga#tacccatt    2062aagcatttcg ccaatcagaa tctcatttta tagtttttcc cattggtctt ta#actaagac    2122tttcttgtag caatctcgta agcagtgaac cccctcagat cagtagaata ta#gtatctgg    2182gggagaagac ttacttcctt cagggcagca gccacagcca ggcttctgtc at#acaggtag    2242atcccgaagc acagagacat aaaaaaggtc tcccagaaaa ctatagacca tt#ctccaagt    2302ggaattccca cttagggctc tggtcactag attgcaacct gtgtgtttgt ca#tcatcctc    2362atctcaccat tgtattgcta tgccctccca taaaaacaca ttgatcccta gc#aagattat    2422tgcattccag attttactgc ctttgctagg cttttgctta gcaaagggct ga#ctttccat    2482tgttatcatg gtgtatatat ttttgtcacc attcccacaa gtatacttga tg#ttgtcata    2542gaacgaacat cctactctat gatttactaa ccaattactt tcccagatca ta#gacctctc    2602tgcatagtag tcataggtct tgactttggg gaaagaaaag gaagctgcag ga#atatttat    2662ctccaaagtc gaatgagaaa gaactccagc aaatccaatg gctacaaact aa#aaatcagc    2722attatttcat attgctgttt cttagctgaa tatggaataa agaactatta tt#ttattttg    2782 aaaaaaaaaa aaaaa               #                  #                   #   2797 <210> SEQ ID NO 4 <211> LENGTH: 529<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4Met Phe Thr Val Leu Thr Arg Gln Pro Cys Gl #u Gln Ala Gly Leu Lys  1               5  #                 10  #                 15Ala Leu Tyr Arg Thr Pro Thr Ile Ile Ala Le #u Val Val Leu Leu Val             20      #             25      #             30Ser Ile Val Val Leu Val Ser Ile Thr Val Il #e Gln Ile His Lys Gln         35          #         40          #         45Glu Val Leu Pro Pro Gly Leu Lys Tyr Gly Il #e Val Leu Asp Ala Gly     50              #     55              #     60Ser Ser Arg Thr Thr Val Tyr Val Tyr Gln Tr #p Pro Ala Glu Lys Glu 65                  # 70                  # 75                  # 80Asn Asn Thr Gly Val Val Ser Gln Thr Phe Ly #s Cys Ser Val Lys Gly                 85  #                 90  #                 95Ser Gly Ile Ser Ser Tyr Gly Asn Asn Pro Gl #n Asp Val Pro Arg Ala            100       #           105       #           110Phe Glu Glu Cys Met Gln Lys Val Lys Gly Gl #n Val Pro Ser His Leu        115           #       120           #       125His Gly Ser Thr Pro Ile His Leu Gly Ala Th #r Ala Gly Met Arg Leu    130               #   135               #   140Leu Arg Leu Gln Asn Glu Thr Ala Ala Asn Gl #u Val Leu Glu Ser Ile145                 1 #50                 1 #55                 1 #60Gln Ser Tyr Phe Lys Ser Gln Pro Phe Asp Ph #e Arg Gly Ala Gln Ile                165   #               170   #               175Ile Ser Gly Gln Glu Glu Gly Val Tyr Gly Tr #p Ile Thr Ala Asn Tyr            180       #           185       #           190Leu Met Gly Asn Phe Leu Glu Lys Asn Leu Tr #p His Met Trp Val His        195           #       200           #       205Pro His Gly Val Glu Thr Thr Gly Ala Leu As #p Leu Gly Gly Ala Ser    210               #   215               #   220Thr Gln Ile Ser Phe Val Ala Gly Glu Lys Me #t Asp Leu Asn Thr Ser225                 2 #30                 2 #35                 2 #40Asp Ile Met Gln Val Ser Leu Tyr Gly Tyr Va #l Tyr Thr Leu Tyr Thr                245   #               250   #               255His Ser Phe Gln Cys Tyr Gly Arg Asn Glu Al #a Glu Lys Lys Phe Leu            260       #           265       #           270Ala Met Leu Leu Gln Asn Ser Pro Thr Lys As #n His Leu Thr Asn Pro        275           #       280           #       285Cys Tyr Pro Arg Asp Tyr Ser Ile Ser Phe Th #r Met Gly His Val Phe    290               #   295               #   300Asp Ser Leu Cys Thr Val Asp Gln Arg Pro Gl #u Ser Tyr Asn Pro Asn305                 3 #10                 3 #15                 3 #20Asp Val Ile Thr Phe Glu Gly Thr Gly Asp Pr #o Ser Leu Cys Lys Glu                325   #               330   #               335Lys Val Ala Ser Ile Phe Asp Phe Lys Ala Cy #s His Asp Gln Glu Thr            340       #           345       #           350Cys Ser Phe Asp Gly Val Tyr Gln Pro Lys Il #e Lys Gly Pro Phe Val        355           #       360           #       365Ala Phe Ala Gly Phe Tyr Tyr Thr Ala Ser Al #a Leu Asn Leu Ser Gly    370               #   375               #   380Ser Phe Ser Leu Asp Thr Phe Asn Ser Ser Th #r Trp Asn Phe Cys Ser385                 3 #90                 3 #95                 4 #00Gln Asn Trp Ser Gln Leu Pro Leu Leu Leu Pr #o Lys Phe Asp Glu Val                405   #               410   #               415Tyr Ala Arg Ser Tyr Cys Phe Ser Ala Asn Ty #r Ile Tyr His Leu Phe            420       #           425       #           430Val Asn Gly Tyr Lys Phe Thr Glu Glu Thr Tr #p Pro Gln Ile His Phe        435           #       440           #       445Glu Lys Glu Val Gly Asn Ser Ser Ile Ala Tr #p Ser Leu Gly Tyr Met    450               #   455               #   460Leu Ser Leu Thr Asn Gln Ile Pro Ala Glu Se #r Pro Leu Ile Arg Leu465                 4 #70                 4 #75                 4 #80Pro Ile Glu Pro Pro Val Phe Val Gly Thr Le #u Ala Phe Phe Thr Val                485   #               490   #               495Ala Ala Leu Leu Cys Leu Ala Phe Leu Ala Ty #r Leu Cys Ser Ala Thr            500       #           505       #           510Arg Arg Lys Arg His Ser Glu His Ala Phe As #p His Ala Val Asp Ser        515           #       520           #       525 Asp<210> SEQ ID NO 5 <211> LENGTH: 3771 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: prim_transcript <222> LOCATION: (921)..(3751)<400> SEQUENCE: 5ccatggcccc ttcattaggg ccccaattgt gactttattg ctcatagtct ct#tccctgcc     60ttggtggctc tcatccccca aacctgaatg cagaagtctt ggtcctagac tc#aactccgt    120gccacccttc agcctacgtt gtgggttcct gctaagctga gcatttacct aa#caatcaag    180acttctgaca gtcctcagtc ctgcccccaa acccccttgg atttctcttt tt#caaggtgg    240tttcggctag gagagtgagc gtggcttggg tgagggcaga tagggtggga gc#atggggca    300tgtatggatg agaccttgac aaagggaccc cggaggaaag acaggggccc tt#tccccctt    360tgtcctggaa acccggctca gccccagccc ttgcccattc tgctgctgct gc#ctggtacc    420ttccacaagg ccagactcct ctccacaaag ctgtggtctg caccagctcc tc#tggctctc    480ctcctctgcc tgctgagggc cgcctcctag cctggctgcc aatcacagga ga#aaggggtt    540gggattttgt ttgtgcctct gtctgagcag agaatggctg ataggcactg ag#cgttgccc    600tggagagccc ctctgtccct gctatcccca tctcccctgg cccagacttc tg#cccttcac    660gcccatccct gaccagcagc cccactcagt ctgggctctg ggtgccagct gt#atagacat    720gccacctgaa cccaggccag agctggtgat gcgtggggct attttaagca ca#gcctcttg    780gcctgcacac tcccctggcc cccagccccc agcagctcag ctactggtca cc#tgccaccg    840cctggaatgc tgattggcag ttggctgggg tgggtggggg ctgggaagac ac#tattataa    900agctgggagt gttgggaagc agccgtcccc gtccagagtc ctctgtggtc cc#tgctgcca    960ccatggccac tcaccgcctc gtgatggtcc ggcacggcga gagcacatgg aa#ccaggaga   1020accgtttctg tggctggttc gatgcagagc tgagtgaaaa ggggaccgag ga#ggccaagc   1080ggggagccaa ggccatcaag gatgccaaga tggagtttga catctgctac ac#gtcagtgc   1140tgaagcgggc catcccgacc ctctgggcca tcctggacgg cacggaccag at#gtggctgc   1200ctgtggtgcg cacttgccgc ttcaatgagc ggcattacgg gggcctcaca gg#cctcaaca   1260aggcagaaac ggccgccaag cacggggagg agcaggtgaa gatctggagg cg#ctccttcg   1320acatcccgcc gcccccgatg gacgagaagc acccctacta caactccatt ag#caaggtgg   1380gctgcctttg ctgggaaggc ctctgggaag ctgcagagtg gggagtcggg tg#ggggccca   1440ctggcttggg agggaaagca gcgtgcctgt gtcccccagg agcgtcggta cg#caggcctg   1500aagcccgggg aactccccac ctgcgagagc ctcaaggaca ccattgcccg gg#ccctgccc   1560ttctggaacg aggagattgt tccccagatc aaggccggca agcgagtgct ca#ttgcagcc   1620cacgggaaca gcctgcgggg cattgtcaag cacctggaag gtaggccacc tt#caggagcc   1680tgggcagggt gggtgggcag cagccagctg gcttctcatc tcagcaaagt ct#ctcgccat   1740gaccagcttt ctagcgtggc tccacatcat tcactgaaaa gaggctgaga ag#ccattttt   1800tagttttgtg aaattttccc catttctgtg taactggaca cactccacag gg#gctgactg   1860cactcgaagc tcgctgtgtc ccgaggtggg gcaggctcca aaggtggcat ct#gccaaggg   1920acacccagct aggaaacgga agggctgggc ttagagcatc tggctccaaa tc#ccaactta   1980ctgtggggcc ctggacaagc cacctccatc tctgggcctc tcccttttcc gg#ggtggtgg   2040ggagctcccc ctggtactga attcctcttg atgtaggctt ggacccctcg ca#gggccctc   2100ccccatcagg tcctcagaat ccctgcatga gcttcaccac ctatctccct ct#ggagcccc   2160tctctgggca aaggaaagac caatcaaaag aggggtgcag gactatggag tg#gccagact   2220ctgggcttgc agctgggctc ccactgaaga gcaagggctg acaaatgggc cc#gggatgca   2280tgggcgcagt aaggcctcgc ccagagtgac tggcacctcc gtccgcctcc ca#ccttagta   2340ttctgacaca agggcagtct aaattagcat ctgaatgacc ttaaagcttg tt#gagtcctg   2400gaaaggctag aagggtgtgc cccagacctc ctgctcctag ggccgttggg ca#gttggcca   2460gagcacccag accggcaggc cccggagacc cagccagccc caagcctgcc cg#ctccaaac   2520acggacacct ggcacctggc actggggcca ggcagaggga aggaccacct gc#ctcctctc   2580ccttccggag acttcatgca gccccatgac cctcccacag cctggtttgg gg#aaagggga   2640cgcacttttg gtggtgaata tgagggattt cactctgact ccccagagaa ca#ttttctta   2700aacccctccc tgcacggagc aggggtggag tggcgcgaac atcaaaggtc ga#gctgctat   2760tcccagctca ggggctgcag gaggcaggca gggtcaggtt tcgaccaggc tc#ggcctccc   2820tgtccctcct ccagctccat tccgcacttg ctcctctgtt caggatgtct ag#aatttaga   2880gcactttaga aacaaagggt gctgggcacg gtggctcact cctgtaatcc ca#gcactttg   2940ggaggctgag gcaggcagat cacctgaggt caggagtttg agaccagcct ga#ccaacatg   3000gtaaaacccg tttctactaa aaatacaaaa ttagccgggt gtggtggcgc tc#acctgtaa   3060tcccagctac ttgggaggct gaggcagaat cacttcaacc caggagatgg ag#gttgcagt   3120gagccaagat cgtgccactg cactccagcc tgggcaagag gagtaaaact cc#atctcaaa   3180aaaaagaaaa agaaaaagaa aagaaaaaaa aaaaccaaag ggtgagtgtc cc#ttcctgac   3240cctcaacttc agtctggctg gagtcacact gggctgaggg aactatggac ag#caccacca   3300cagatcacag ccacttgggt ggggctgaag tccccatttt tttcaccact gg#gctatttc   3360tgtaggctgc ttggtctaac tcagttactc cttgaccttt ggcaacattt ct#gtggcctc   3420gttctcaggg ctgggaagga attggtgcca ggggaactgg ctctgtggac ca#taaaggtc   3480acatagtgtc tgctgtgtaa acaggctggg gacagagggg ctaaggacac ct#attccttc   3540cggcataggg atgtcagacc aggcgatcat ggagctgaac ctgcccacgg gg#atccccat   3600tgtgtatgag ctgaacaagg agctgaagcc caccaagccc atgcagttcc tg#ggtgatga   3660ggaaacggtg cggaaggcca tggaggctgt ggctgcccag ggcaaggcca ag#tgaggggt   3720gggcttgggc aataaaggca cctcccccaa cagcctggag tctccagcgc a #           3771 <210> SEQ ID NO 6 <211> LENGTH: 992 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (242)..(583) <400> SEQUENCE: 6ttttgtgcga gagccgcagc gccgcctctt ctctcgcgcc ctcgcctctt cc#tccgcctc     60ctccttcgcc tcttcctgcc tcctcccggc ttccgccgcc gccactccag cc#taatccca    120accccagggc gaaggttttc ttatttattt ccgttttctc gccactacag cc#tcctgaca    180aggtgatccg ggcgggcccc gcaggaattt tatcccctca ccggcctcac ac#tagtatcg    240 c atg tcc act atc cag aac ctc caa tct ttc #gac ccc ttt gct gat gca    289   Met Ser Thr Ile Gln Asn Leu Gln Ser P#he Asp Pro Phe Ala Asp Ala     1               # 5                 # 10                  # 15 act aag ggt gac gac tta ctc ccg gca ggg ac#t gag gat tac att cat      337Thr Lys Gly Asp Asp Leu Leu Pro Ala Gly Th #r Glu Asp Tyr Ile His             20      #             25      #             30ata aga atc cag caa cgg aac ggc aga aag ac#a ctg act act gtt cag      385Ile Arg Ile Gln Gln Arg Asn Gly Arg Lys Th #r Leu Thr Thr Val Gln         35          #         40          #         45ggc att gca gat gat tat gac aaa aag aaa ct#t gtg aaa gct ttc aaa      433Gly Ile Ala Asp Asp Tyr Asp Lys Lys Lys Le #u Val Lys Ala Phe Lys     50              #     55              #     60aag aaa ttt gcc tgt aat ggt act gtg att ga#a cat cct gaa tac gga      481Lys Lys Phe Ala Cys Asn Gly Thr Val Ile Gl #u His Pro Glu Tyr Gly 65                  # 70                  # 75                  # 80gag gtt att cag ctt caa ggt gac caa aga aa#a aac atc tgc cag ttt      529Glu Val Ile Gln Leu Gln Gly Asp Gln Arg Ly #s Asn Ile Cys Gln Phe                 85  #                 90  #                 95ctc ttg gag gtt ggc att gta aag gag gaa ca#g ctt aag gtt cat gga      577Leu Leu Glu Val Gly Ile Val Lys Glu Glu Gl #n Leu Lys Val His Gly            100       #           105       #           110ttc taa aatgaaccta aatacgtgga gaatttcttg aatagttttg tt#ctctaaac       633 Pheccggtttggc tgccttgtga aatgattccc tgcagtaaac ggacttttca tt#tatttaat    693cattcaaact tccattcaca tctgcatgat tacagaaaac atggggtatg ta#gactagta    753acacataaga aaattgcagt aagatggtaa caaaacctca tattgtcttt ac#atgtttcc    813aatggaaaat gttttgagtg tttattgttc agtttattac gtttcacttg at#taaatttt    873ttttgttgtt gtattaaacc atgtacgttg cagcttaaca ataaaaaaaa aa#tctatgaa    933tctttgtgag caattatgct cccaaatcta agcaagtaat aaagaagggg ga#ttcaaag     992 <210> SEQ ID NO 7 <211> LENGTH: 113 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7Met Ser Thr Ile Gln Asn Leu Gln Ser Phe As #p Pro Phe Ala Asp Ala  1               5  #                 10  #                 15Thr Lys Gly Asp Asp Leu Leu Pro Ala Gly Th #r Glu Asp Tyr Ile His             20      #             25      #             30Ile Arg Ile Gln Gln Arg Asn Gly Arg Lys Th #r Leu Thr Thr Val Gln         35          #         40          #         45Gly Ile Ala Asp Asp Tyr Asp Lys Lys Lys Le #u Val Lys Ala Phe Lys     50              #     55              #     60Lys Lys Phe Ala Cys Asn Gly Thr Val Ile Gl #u His Pro Glu Tyr Gly 65                  # 70                  # 75                  # 80Glu Val Ile Gln Leu Gln Gly Asp Gln Arg Ly #s Asn Ile Cys Gln Phe                 85  #                 90  #                 95Leu Leu Glu Val Gly Ile Val Lys Glu Glu Gl #n Leu Lys Val His Gly            100       #           105       #           110 Phe<210> SEQ ID NO 8 <211> LENGTH: 445 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 8caccatgtcc actatccaga acctccaatc tttcgacccc tttgctgatg ca#actaaggg     60tgacgactta ctcccggcag ggactgagga ttacattcat ataagaatcc ag#caacggaa    120cggcagaaag acactgacta ctgttcaggg cattgcagat gattatgaca aa#aagaaact    180tgtgaaagct ttcaaaaaga aatttgcctg taatggtact gtgattgaac at#cctgaata    240cggagaggtt attcagcttc aaggtgacca aagaaaaaac atctgccagt tt#ctcttgga    300ggttggcatt gtaaaggagg aacagcttaa ggttcatgga ttcaagggcg ag#cttcgagg    360tcacccattc gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ct#acgcgtac    420 cggtcatcat caccatcacc attga          #                   #              445 <210> SEQ ID NO 9<211> LENGTH: 146 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence:  Synthetic       Peptide <400> SEQUENCE: 9Met Ser Thr Ile Gln Asn Leu Gln Ser Phe As #p Pro Phe Ala Asp Ala  1               5  #                 10  #                 15Thr Lys Gly Asp Asp Leu Leu Pro Ala Gly Th #r Glu Asp Tyr Ile His             20      #             25      #             30Ile Arg Ile Gln Gln Arg Asn Gly Arg Lys Th #r Leu Thr Thr Val Gln         35          #         40          #         45Gly Ile Ala Asp Asp Tyr Asp Lys Lys Lys Le #u Val Lys Ala Phe Lys     50              #     55              #     60Lys Lys Phe Ala Cys Asn Gly Thr Val Ile Gl #u His Pro Glu Tyr Gly 65                  # 70                  # 75                  # 80Glu Val Ile Gln Leu Gln Gly Asp Gln Arg Ly #s Asn Ile Cys Gln Phe                 85  #                 90  #                 95Leu Leu Glu Val Gly Ile Val Lys Glu Glu Gl #n Leu Lys Val His Gly            100       #           105       #           110Phe Lys Gly Glu Leu Arg Gly His Pro Phe Gl #u Gly Lys Pro Ile Pro        115           #       120           #       125Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Th #r Gly His His His His    130               #   135               #   140 His His 145<210> SEQ ID NO 10 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Synthetic      Primer <400> SEQUENCE: 10 caccatgtcc actatccaga acctcc          #                   #              26 <210> SEQ ID NO 11<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence:  Synthetic       Primer <400> SEQUENCE: 11gaatccatga accttaagct gttc           #                   #            24

What is claimed is:
 1. A method for identifying a subject at risk forthe development of non-small cell lung cancer comprising: (a) obtaininga test sample from a subject; (b) providing an RPL14 gene probe fromhuman chromosomal region 3p and a 10q22 DNA gene probe; (c) contactingsaid probes with said test sample; and (d) analyzing DNA from said testsample for loss of heterozygosity in RPL14 and 10q22, whereby loss ofRPL14 and 10q22 heterozygosity indicates risk for the development ofnon-small cell lung cancer.
 2. The method of claim 1, wherein said testsample comprises a surgical or biopsy specimen, a paraffin embeddedtissue, a frozen tissue imprint, a sputum, esophageal brush, a fineneedle aspiration, a buccal smear or a bronchial lavage.
 3. The methodof claim 1, further comprising providing a GC20 gene probe from humanchromosomal region 3p and performing steps (c) and (d) with said GC20gene probe.
 4. The method of claim 1, wherein said subject is a smoker.5. The method of claim 1, wherein said subject is a former smoker. 6.The method of claim 1, wherein said subject is a non-smoker.
 7. Themethod of claim 1, wherein said test sample comes from said subject whohas not previously been diagnosed with cancer.
 8. The method of claim 1,wherein said probes are labeled with fluorophores.
 9. The method ofclaim 1, wherein one of said probes is labeled with digoxigenin.
 10. Themethod of claim 1, wherein size of said probes is between 1000 and 2000base pairs.
 11. The method in claim 1, further comprising performing aspiral CT-scan.
 12. The method of claim 1, further comprisingadministering to said subject chemopreventive drugs, nutritionalsupplements, chemotherapeutic drugs or biological modifying responsedrugs.
 13. The method of claim 1, wherein said method is used toidentify subjects who need an intensive follow-up protocol.
 14. Themethod of claim 1, wherein said probes are used to identify subjects whoare suitable for novel investigational therapeutic approaches.
 15. Themeted of claim 1, wherein a control probe is used.
 16. The method ofclaim 15, wherein said control probe is labeled with a fluorophore. 17.The method of claim 16, wherein said control probe is labeled withspectrum orange.
 18. The meted of claim 15, wherein said control probeis a chromosome 3 stable marker.
 19. The method of claim 15, whereinsaid control probe is Centromere 3 (CEP 3).
 20. The method of claim 1,wherein analyzing comprises using FISH.
 21. The method of claim 1,wherein said probes are used as a biomarkers for the early detection ofearly neoplastic events or cancer.